Substrate for immobilizing functional substances and method for preparing the same

ABSTRACT

A substrate having compounds disposed thereon for immobilizing a functional molecule, each compound having a chain including: a moiety R that is chemically coupled to the substrate, the moiety R being selected from the group consisting of an ether, ester, carbonyl, carbonate ester, thioether, disulfide, sulfinyl, sulfonyl, and carbonothioyl; and an epoxide-containing moiety that is coupled to the moiety R by a linker including at least one nucleophilic group. Methods of preparing the substrate and use of the substrate are also disclosed

TECHNICAL FIELD

The present invention generally relates to a substrate forimmobilization of functional substances. The present invention alsorelates to methods of preparing the substrate.

BACKGROUND

Biological and chemical assays and processes that are used analyticallyor preparatively for research, clinical, diagnostic and industrialpurposes often require fixation, or immobilization, of a functionalsubstance onto a solid support (or substrate). This fixation oftenimproves the stability and versatility of the substance withoutcompromising its effectiveness and activity, and enables repeated usageof the substance. For example, functional substances which includebiological substances such as enzymes, are typically immobilized on aninert support like silica or polyacrylamide gel to improve theirstability against changing pH or temperature conditions when used inenzyme-catalyzed industrial processes, and to facilitate theirsubsequent separation from the reaction products. This enables re-use ofthe immobilized enzymes and significantly facilitates productpurification, which leads to more cost-effective processes.

Immobilization of a functional substance including a biologicalsubstance may be effected by physical immobilization or chemicalimmobilization. One form of physical immobilization is physicaladsorption (physisorption), where the functional or biological substanceis attached to the substrate via encapsulation or electrostatic,hydrophobic or Van der Waals forces. Whilst physical adsorption providesa relatively simple immobilization method with wide applicability to awhole range of functional and/or biological substances, it often doesnot provide a sufficiently stable immobilization and is susceptible toleaching of the immobilized functional and/or biological substances.

A more stable method of immobilization of functional and/or biologicalsubstances is chemical immobilization, which covalently binds thefunctional and/or biological substance to the substrate as a result of achemical reaction. Chemical immobilization typically results in improvedactivity, reduced non-specific adsorption, and higher stability of thefunctional and/or biological substance. However, chemical immobilizationgenerally requires the chemical modification of the functional and/orbiological substance or the substrate to promote their efficientbinding.

Modification of the surface of a solid support material, or“pre-activation” of a solid support, to improve its binding to afunctional and/or biological substance, typically involves theincorporation of reactive chemical moieties onto the surface of thegenerally poorly reactive polymeric material. Surface modification canbe achieved by physical means, such as non-covalent attachment of anaffinity spacer, or chemical means such as glutaraldehyde activation,cyanogen bromide activation, bromoacetylation, diazotation,ionizing-radiation induced oxidation and chemical grafting.

The non-covalent attachment of an affinity spacer is, however,associated with poor reproducibility and/or unstable binding to thesurface of the substrates. Some covalent attachments, most noteworthyimines, but to a lesser extent also esters, can be hydrolyzed under thereaction conditions used for enzymatic reactions, resulting in partialloss of immobilized enzyme and leakage of enzyme into the reactionmedium. Such problems may affect, amongst others, immobilization methodsbased on glutaraldehyde activation and bromoacetylation. Whilstdiazotation, cyanogen bromide activation, ionizing-radiation inducedoxidation, and chemical grafting produce covalent bonds which are morestable than non-covalent bonds, these methods involve the use ofhazardous, expensive, complicated, and/or harsh reaction conditions.

Some of these methods also result in a high net charge on the solidsupport, which causes undesirable non-specific electrostatic binding ofthe functional and/or biological substance during subsequent proceduresin a biological/chemical process. Another common problem encounteredwith the use of harsh reaction conditions is the unfavorablemodification of surface properties, which may hamper the attachment of afunctional and/or biological substance, particularly a large polymericsubstance. This can lead to low loading of the functional and/orbiological substance onto the substrate. Yet other problems encounteredwith some commercially available activated solid supports are lowstability, pronounced toxicity and a lack of biocompatibility, resultingin short shelf life, difficult handling, and limited applicability formedical purposes.

Some of these methods rely on the further modification of“pre-activated” supports with an epoxysilane coupling agent for theimmobilization of hydrophilic molecules. Other methods rely onpreparation of a substrate by reacting a bisepoxyoxirane linker toimmobilize a molecule to the substrate. The aliphatic linkers used inthese methods lead to a decrease in the amount of reactive groupsavailable for immobilization, a decrease in biocompatibility and adecrease in reproducibility.

There is a need to provide methods of preparing a substrate forimmobilization of functional and biological substances that overcome, orat least ameliorate, one or more of the disadvantages described above.

There is a need to provide methods that are convenient, inexpensive,robust, and reliable for preparing a substrate for immobilization offunctional and biological substances.

There is also a need to provide substrates which are stable, easy tohandle, inexpensive, non-toxic, biocompatible and bio-degradable forimmobilization of functionally and biologically active substances, andwhich can be used for immobilization of a wide range of substances athigh loading densities with improved activity and reactivity.

SUMMARY

According to a first aspect of the invention, there is provided asubstrate having compounds disposed thereon for immobilizing afunctional molecule, each compound having a chain comprising: a moiety Rthat is chemically coupled to the substrate, said moiety R beingselected from the group consisting of an ether, ester, carbonyl,carbonate ester, thioether, disulfide, sulfinyl, sulfonyl, andcarbonothioyl; and an epoxide-containing moiety that is coupled to themoiety R by a linker comprising at least one nucleophilic group.

In one embodiment, the substrate comprises an additional epoxidecontaining group coupled to the chain. In another embodiment the numberof additional epoxide containing group is selected from the number 1, 2,3, 4 and 5. In one embodiment, the linker comprises additionalnucleophilic groups to which said additional epoxide containing groupsare coupled to said chain. This is advantageous, as the density of theepoxide-containing groups available to react with a functional moleculeis increased and consequently the number of immobilization sites thatare available for immobilizing a substance is also increased.

It is an advantage of the disclosure that the linker increases thelength of the tether between the functional molecule and the substrateand aids in the binding of the functional molecule to the substrate.

In another embodiment, the linker comprises a di-nucleophilic species.In one embodiment, the di-nucleophilic linker is selected from at leastone of an alkyl-diamine and an alkene-diamine. Advantageously, thediamine linker may introduce additional sites for epoxy-activation.Without being bound by theory, it is believed that up to five moleculesof epoxide-containing compound (such as epichlorohydrin) can react withone molecule of diamine linker. This is advantageous as this permits anincrease in the density of, for example, an epoxide-containing compoundand consequently the number of immobilization sites that are availablefor immobilizing a functional molecule.

In another embodiment, the linker comprises a polynucleophilic species.In another embodiment, the polynucleophilic species may be a polyaminesuch as putrescine, spermidine, spermine, cadaverine, diethylenetriamine, triethylene tetramine, tetraethylene pentamine, andtetrahydrofurfuryl amine.

The introduction of the amine groups of the linker also beneficiallyserves as an internal pH buffer for the immobilized substance. It is afurther advantage that amine groups can also function as ion-exchangersto provide stabilizing conditions for the immobilized substance. It is afurther advantage that amine groups are strongly nucleophilic making thecoupling of the first and second epoxide-containing compound moreefficient than other nucleophilic groups, for example OH groups. Thestrongly nucleophilic nature of the amine groups of the linker isfurther advantageous as this permits the use of long linkers whilstmaintaining reactivity with the epoxide-containing compound. This isfurther advantageous as the use of longer linkers permits a furtherreduction of steric hindrance between the substrate and the immobilizedsubstance. More advantageously, diamines such as hexanediamine arerelatively cheap as compared to other linkers and are commerciallyavailable commodity materials. The low cost of hexanediamine ensuresthat the disclosed substrate can be mass-produced at relatively lowcosts.

It is a further advantage that the alkyl-amine bond formed between theepoxide-containing compound and the hexanediamine linker is resistant tohydrolysis under physiological conditions such that it can be used inaqueous systems, for example dialysis devices. It is a further advantagethat the substrate in accordance with the disclosure is biologicallyinert.

According to a second aspect, there is provided a method of immobilizinga functional molecule on a substrate comprising the step of exposing thefunctional molecule to the substrate according to the disclosure.

According to a third aspect there is provided a method of preparing asubstrate for immobilization of functional molecules thereon, the methodcomprising the steps of: (i) providing electrophilic compounds coupledto the surface of the substrate; (ii) allowing the electrophiliccompounds to undergo a nucleophilic substitution reaction to provide anucleophilic group thereon and thereby increase the nucleophilicity ofthe substrate surface; (iii) allowing the nucleophilic group to undergoa nucleophilic substitution reaction with another electrophilic compoundto provide an electrophilic group on the substrate surface and therebyincrease the electrophilicity of the substrate.

It is an advantage of the method that the elongated spacer attached tothe electrophilic groups of the substrate provides increasedaccessibility of the functional compound to the electrophilic group andconcomitantly permits an increase in the density and reactivity of theelectrophilic group to the functional molecule to thereby immobilize thefunctional molecule on the substrate. It is a further advantage thatstep (iii) of the method also provides increased accessibility of afunctional molecule to the electrophilic group for subsequentimmobilization on the substrate.

In one embodiment, steps (ii) and (iii) are repeated n number of timesto form n generations of electrophilic groups on said substrate. This isadvantageous as this permits an elongation of the spacer and an increasein the density of the electrophilic groups and consequently the numberof immobilization sites that are available for immobilizing a functionalmolecule to the substrate.

It is a further advantage that step (iii) of the method permits arelatively faster reaction between the linker and the secondelectrophilic compound. This results in a decreased rate of hydrolysisof the electrophilic compounds and a higher incorporation ofnon-hydrolysed electrophilic groups on to the substrate. This is afurther advantage, as this also permits an increase in the density ofthe electrophilic groups on the substrate for immobilizing a functionalmolecule.

It is a further advantage of the method that the electrophilic groupsare displaced relative to the substrate such that the ability of thefunctional molecule to be immobilized thereon is enhanced relative tohaving a substrate with only one electrophilic compound being directlycoupled to a substrate. It is a further advantage that this relativedisplacement also permits increased accessibility of a functionalmolecule to the electrophilic group for subsequent immobilization on thesubstrate.

According to a fourth aspect, there is provided a sorbent cartridge foruse in a dialysis device, the sorbent cartridge comprising a substrateas described herein for immobilizing urease.

According to a fifth aspect, there is provided a dialysis methodcomprising the steps of: exposing a dialysate containing urea to asubstrate as described herein; and removing the dialysate from saidsubstrate.

According to a sixth aspect, there is provided a dialyzer for use in adialysis device, the dialyzer comprising a substrate as described hereinfor immobilizing urease. Hence, urease may be immobilised onto adialysis membrane such as a cellulose acetate membrane filter comprisedwithin the dialyser.

According to a sixth aspect, there is provided a method of modifying adialysis membrane for immobilizing functional molecules thereon, themethod comprising the steps of:

-   -   (i) coupling electrophilic compounds to the membrane surface;    -   (ii) allowing the electrophilic compounds to undergo a        nucleophilic substitution reaction to provide a nucleophilic        group thereon and thereby increase the nucleophilicity of the        membrane surface; and    -   (iii) allowing the nucleophilic group to undergo a nucleophilic        substitution reaction with another electrophilic compound to        provide an electrophilic group on the membrane surface and        thereby increase the electrophilicity of the membrane surface        for immobilizing functional molecules thereon.

In one embodiment, the membrane is a cellulose acetate membrane.

Advantageously, the method can be used to modify an off-the-shelfdialysis membrane, such as a cellulose acetate membrane, of a dialyzer.This modification step allows the surface of the dialysis membrane tohave an increased ability to immobilize functional molecules, such asdialysate enzymes, thereon when used in a dialyzer.

According to a eighth aspect, there is provided the use of the substrateas described herein in a dialysis device.

In one embodiment, there is provided a method of preparing a substratefor immobilization of functional substances thereon, the methodcomprising the steps of chemically coupling a first electrophiliccompound to the substrate; and chemically coupling a secondelectrophilic compound to the first electrophilic compound that has beencoupled to the substrate, wherein said second electrophilic compound,when coupled to said first electrophilic compound, is configured toimmobilize the functional substance thereon.

Advantageously, the first and second electrophilic compounds areselected to be displaced relative to each other such that the ability ofthe functional substance to be immobilized thereon is enhanced relativeto having a substrate with only one electrophilic compound being coupledto a substrate. More advantageously, the first and second electrophiliccompounds are selected to be displaced relative to each other such thatsteric hindrance effects in the neighborhood of the second electrophileare reduced or minimized to enhance binding of the functional substanceto the second electrophilic compound in use.

More advantageously, the first and/or second electrophilic compound maybe a di-electrophile that effectively converts a poorly nucleophilicsubstrate into a strongly electrophilic substrate. This constitutes achange in polarity and reactivity of the substrate.

More advantageously, the method is a simple and cost efficient way toproduce a substrate that has a relatively high reactivity to functionalsubstances for immobilization thereon as compared to substrates producedby known methods. More advantageously, the second electrophilic compoundis capable of binding stably to a functional substance including abiological substance such as an enzyme. Even more advantageously, thesecond electrophilic compound offers a binding site for the functionalsubstance that is at an appropriate distance away from substrate suchthat steric hindrance is reduced. In one embodiment, the density ofelectrophilic groups per gram of the substrate is from about 0.1 toabout 1 mmol/g.

This in turn reduces impediment during the immobilization of thefunctional substance and allows the functional substance to be anchoredto the substrate easily, via the second epoxide containing compound. Italso enhances the accessibility and structural flexibility of the boundsubstance (enzyme), thereby increasing its activity. The disclosedmethod is also capable of producing substrates that can immobilizechiral ligands, affinity ligands and/or ion exchange particles.

In one embodiment, the first and second electrophilic compounds areepoxide containing compounds. In one embodiment, the disclosed methodcomprises the step of using a linker to couple the secondepoxide-containing compound to the first epoxide containing compound.This increases the length of the tether between the active oxirane siteand the substrate and aids in the binding of the functional substance tothe oxirane site. It also enhances the accessibility and thereby theactivity of the bound substance such as an enzyme. Advantageously, thelinker may contain additional functional groups to impart desirouschemical properties to the substrate. For example, the linker maycontain amine groups that have buffering properties which may bebeneficial when the substrate is used in applications such as dialysisdevices. The linker may also contain groups that can function asanti-oxidants or metal scavengers which supplements the functions of thesubstrate in certain applications. More advantageously, the linker mayalso provide increased sites for binding of the second electrophiliccompound and/or subsequent epoxide-containing compounds. In effect, thelinker may increase the number of epoxide-containing compounds coupledto the substrate which in turn increases the probability and strength ofimmobilization of the functional substance. The linker may also beneutral and inert which does not adversely affect the functional orbiological property of a functional or biological substance coupled toit.

In one embodiment, the linker does not contain an epoxide group. Thefunctional linker may also comprise a nucleophilic group.Advantageously, the nucleophilic group is reactive and capable ofchemically binding to the electrophilic (epoxide-containing) compounds.In one embodiment, the functional linker is a di-nucleophilic linker.The presence of two nucleophilic groups allows the linker to bind toboth the first and second epoxide-containing compounds, forming a bridgebetween the two epoxide-containing compounds. In one embodiment, atleast one of the nucleophiles of the di-nucleophilic linker is selectedfrom the group consisting of NH, NR, NHO, NRO, O, S, Se, COO, CONH,CONR, CSS, COS, CONHO, CONRO, CONHNH, CONRNH, CONR¹NR², CNO, Ph and PR,

where R, R¹ and R² are independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl.

The di-nucleophilic linker may also have the general formula (I):

wherein:

X and Y are independently selected from NH, NR, NHO, NRO, O, S, Se, COO,CONH, CONR, CSS, COS, CONHO, CONRO CONHNH, CONRNH, CONRNR, CNO, PH andPR;

R is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl and optionallysubstituted heteroaryl; and

n is an integer from 0 to 25.

In another embodiment, the di-nucleophilic linker has the generalformula (II):

wherein:

X and Y are independently selected from NH, NR, NHO, NRO, O, S, Se, COO,CONH, CONR, CSS, COS, CONHO, CONRO CONHNH, CONRNH, CONRNR, CNO, PH, PR;

where R, R¹, R², R³, R⁴ are independently selected from the groupconsisting of hydrogen, optionally substituted alkyl, optionallysubstituted alkenyl, optionally substituted alkynyl, optionallysubstituted aryl and optionally substituted heteroaryl; and

m, n, p and q is an integer independently selected from 0 to 25.

In one embodiment, the di-nucleophilic linker is an alkyl-diamine suchas hexanediamine or ethylene diamine. Advantageously, the diamine linkermay introduce additional sites for epoxy-activation. Without being boundby theory, it is believed that up to five molecules ofepoxide-containing compound (such as epichlorohydrin) can react with onemolecule of diamine linker. This is advantageous as this permits anincrease in the density of, for example, an epoxide-containing compoundand consequently the number of immobilization sites that are availablefor immobilizing a substance. The introduction of the amine groups ofthe linker also beneficially serves as an internal pH buffer for theimmobilized substance. It is a further advantage that amine groups canalso function as ion-exchangers to provide stabilizing conditions forthe immobilized substance. It is a further advantage that amine groupsare strongly nucleophilic making the coupling of the first and secondepoxide-containing compound more efficient than other nucleophilicgroups. The strongly nucleophilic nature of the amine groups of thelinker is further advantageous as this permits the use of long linkerswhilst maintaining reactivity with the epoxide-containing compound. Thisis further advantageous as the use of longer linkers permits a furtherreduction of steric hindrance between the substrate and the immobilizedsubstance. More advantageously, the hexanediamine is relatively cheap ascompared to other linkers and is a commercially available commoditymaterial. The low cost of hexanediamine ensures that the disclosedsubstrate can be mass-produced at relatively low costs. It is a furtheradvantage that the alkyl amine bond formed when using a hexanediaminelinker is resistant to hydrolysis under physiological conditions suchthat it can be used in aqueous systems, for example dialysis devices. Itis a further advantage of the hexanediamine linker that it isbiocompatible and biodegradable.

In one embodiment, at least one of the electrophilic compound and thesecond electrophilic compound is an epihalohydrin. Preferably, theepihalohydrin is epichlorohydrin. Similarly, epichlorohydrin isrelatively cheap as compared to other epihalohydrins and is procurableeasily as it is a commercially available commodity material. Again, thelow cost of epichlorohydrin ensures that the disclosed substrate can bemass-produced at relatively low costs. Epichlorohydrin also tends toreact very rapidly and exhaustively, only giving non-toxic products(glycerol and amino-glycerols) and thus is suitable for use in preparinga substrate that would eventually be used for medical applications.Moreover, because epichlorohydrin is partly miscible with water andfully miscible with alcohol, any excess epichlorohydrin can berelatively easily removed by washing the substrate with water and/oralcohol. Furthermore, epichlorohydrin and its hydrolysis products arevolatile and can therefore be efficiently removed by evaporation.

In one embodiment, the method comprises selecting a poorly reactivesubstrate. The substrate of the disclosed method may be a bead,micro-sized particle, nanosized particle, a membrane, a mesh, a scaffoldor any solid support that is capable of being prepared using thedisclosed method to immobilize a functional substance including abiological substance thereon. In one embodiment, the substrate isselected from the group consisting of a polyester substrate, a polyamidesubstrate, an epoxy resin substrate, a polyacrylate substrate, ahydroxyl-functionalized substrate and a polysaccharide-based substrate.The polysaccharide-based substrate may be selected from the groupconsisting of cotton linters, cotton pulp, cotton fabrics, cellulosefibers, cellulose beads, cellulose powder, microcrystalline cellulose,cellulose membranes, rayon, cellophane, cellulose acetate, celluloseacetate membranes, chitosan, chitin, dextran derivatives and agarosederivatives. The substrates may also be biodegradable and thusenvironmentally friendly, which allows their application inenvironmentally sensitive applications such as agricultural applicationsor waste treatment applications. The substrate may also be biocompatiblesuch that when the substrate is implanted into the human body or inconjunction with the human body, for example in dialysis, little or noadverse health effects are elicited.

The chemical coupling steps of the disclosed methods may be undertakenat a temperature range of from −30° C. to 100° C., preferably from 0° C.to 100° C. In one embodiment, the step of chemically coupling a firstelectrophilic compound to the substrate is carried out at a temperaturefrom about 50° C. to 60° C.; the step of chemically coupling a linker tothe first electrophilic compound is carried out at a temperature fromabout 20° C. to 40° C.; the step of chemically coupling a secondelectrophilic compound to the linker is carried out at a temperaturefrom about 20 to 40° C.; and the step of chemically coupling thefunctional substance to the second electrophilic compound is carried outat a temperature from about 2 to 6° C. Advantageously, the substrate canbe produced and/or prepared at mild conditions for example at roomtemperature, and in normal atmosphere. This again translates to lowerproduction costs and increased ease of handling. More advantageously,the final immobilization reaction can be carried out under very mildconditions, such as in aqueous buffer at 2 to 6° C. and normalatmosphere, and does not require any additional reagents. Thiseliminates the risk of deactivation or denaturation of the bioactivesubstance by extreme conditions or strong reagents, such as might beproblematic in other immobilization methods. Even more advantageously,as the method can be carried out at ambient temperatures, theimmobilization of bioactive substances on the substrate might also becarried out simultaneously or subsequent to the activation of thesubstrate.

In one embodiment, the functional substances are biologically activesubstances such as enzymes, for example urease. Advantageously, whenurease is immobilized on the substrate, the substrate containing theimmobilized urease can be used for dialysis applications, for examplefor the regeneration of peritoneal dialysate or hemodialysate. Theenzymes may also be at least one of uricase, creatininase, lipase,esterase, cellulase, amylase, pectinase, catalase, acylase, penicillinamidase, proteinase-K.

In one embodiment, the disclosed method further comprises the step ofchemically coupling one or more subsequent electrophilic compounds toboth the first and second electrophilic compounds, wherein saidsubsequent electrophilic compound(s), when coupled to both said firstand second electrophilic compounds is/are configured to immobilize thefunctional substance thereon. For example, a third, fourth, fifth, sixthelectrophilic compound and so on may be coupled to both the first andsecond electrophilic compounds. The electrophilic groups as disclosedherein may contain at least one epoxide group.

In another embodiment, there is provided a method of immobilizing afunctional molecule on a substrate, the method comprising the steps ofproviding the substrate having compounds thereon prepared by the methodof the disclosure, each of said compounds comprising an ether-containingmoiety that is chemically coupled to the substrate and anepoxide-containing moiety that is coupled to the ether moiety; andintroducing a solution containing said functional molecule to saidcompounds disposed on said substrate, wherein the epoxide-containingmoiety forms a chemical bond with said functional molecule to immobilizeit thereto.

Advantageously, this chemical bond may be a non-hydrolyzable covalentbond, such as an amine-bond. Consequently, the functional molecule willbe immobilized on the substrate with sufficient stability and cannot beeasily removed from the substrate.

The method of the second aspect may further comprise the step ofapplying a substantially homogenous mixture of stabilizing additivesonto the surface of the substrate to stabilize said functional molecule.In one embodiment, the step of applying the substantially homogenousmixture of additives comprises evaporating the solvent of a solution ofsaid additives onto the substrate. The stabilizing additives may beselected from the group consisting of a sugar such as glucose, anorganic acid such as ethylenediaminetetraacetic acid, an amino acid suchas cysteine and a sugar acid such as ascorbic acid and thiols such asmercaptoethanol. In another embodiment, there is provided a substratehaving compounds disposed thereon for immobilizing a functionalmolecule, each compound comprising an ether-containing moiety that ischemically coupled to the substrate and an epoxide-containing moietythat is coupled to the ether moiety by a linker comprising at least onenucleophilic group, whereby said epoxide-containing moiety is disposedfrom said ether-containing moiety to immobilize the functional moleculeto said epoxide-containing moiety without substantial steric hindrancebeing caused by said ether containing moiety or the substrate.Advantageously, the substrate has an improved stability and can beproduced at a relatively low cost when compared to known substrates thatcan effectively immobilize functional substances. More advantageously,the substrate can be reused repeatedly without substantially losing itsenzymatic properties, due to the high stability of bonding between thebiomolecule and the epoxide-containing moiety. In addition, as there isno leaching of potentially hazardous substances, the substrate issuitable for use in medical applications such as for peritonealdialysis.

In one embodiment, the linker is a non-hydrocarbon such as hydrazine,hydroxylamine, ammonia, water, or hydrogen sulfide.

In one embodiment, the linker is a saturated or unsaturated aliphaticchain having from 2 to 18 carbon atoms, 2 to 16 carbon atoms, 2 to 14carbon atoms, 2 to 12 carbon atoms, or 2 to 10 carbon atoms, 2 to 8carbon atoms, 2 to 6 carbon atoms, and 2 to 4 carbon atoms. In oneembodiment, the linker is a saturated aliphatic chain having 4 to 8carbon atoms, more preferably 6 carbon atoms. The nucleophilic group ofsaid linker may be located at one of the terminal ends of the aliphaticchain or in between the terminal ends of the aliphatic chain. In oneembodiment the nucleophilic group of said linker may be chemicallycoupled to the aliphatic chain by way of a branch chain extendingtherefrom. In one embodiment, there are two nucleophilic groups disposedon said linker, preferably at terminal ends of the aliphatic chain. Inone embodiment at least one nucleophilic group is disposed on a terminalend of the aliphatic chain and is coupled to either the ether orepoxide-containing moiety with a secondary aliphatic linker chaintherebetween. The secondary aliphatic linker chain may have from 1 to 3carbon atoms.

The substrate may further comprise a coating disposed on said substrate,the coating comprising a substantially homogenous mixture of stabilizingadditives. The stabilizing additives may be selected from the groupconsisting of a sugar such as glucose, an organic acid such asethylenediaminetetraacetic acid, an amino acid such as cysteine and asugar acid such as ascorbic acid.

In another embodiment, there is provided a sorbent cartridge for use ina dialysis device, the sorbent cartridge comprising a substrate havingcompounds disposed thereon that comprise an immobilized urease, eachcompound comprising an ether-containing moiety that is chemicallycoupled to the substrate and an epoxide-containing moiety that iscoupled to the ether moiety by a linker comprising at least onenucleophilic group, whereby said epoxide-containing moiety is disposedfrom said ether-containing moiety to immobilize the urease molecule tosaid substrate without substantial steric hindrance being caused by saidether containing moiety or the substrate.

In another embodiment, there is provided a dialysis method comprisingthe steps of exposing a dialysate containing urea to a substrate havingcompounds disposed thereon that comprise an immobilized urease, eachcompound comprising an ether-containing moiety that is chemicallycoupled to the substrate and an epoxide-containing moiety that iscoupled to the ether moiety by a linker comprising at least onenucleophilic group, whereby said epoxide-containing moiety is disposedfrom said ether-containing moiety to immobilize the urease molecule tosaid substrate without substantial steric hindrance being caused by saidether containing moiety or the substrate; and removing the dialysatefrom said substrate after at least a portion of said urea has beenbroken down.

In another embodiment, there is provided the use of the substrateaccording to the disclosure in a dialysis device. Advantageously, thesubstrate can be used to remove toxins from the dialysate in thedialysis device effectively and safely.

Definitions

The following words and terms used herein shall have the meaningindicated:

The term “epoxide”, “epoxy group” or “oxirane” depicts a chemicalfunctional group consisting of a three-membered ring arrangement of twocarbon atoms and one oxygen atom. The two carbon atoms in thethree-membered ring may be independently substituted. The term “epoxide”may also depict a molecule or compound that comprises at least one epoxygroup.

The term “epoxide-containing compound” means any compound that is anepoxide or a compound which contains an epoxide moiety. Exemplaryepoxide containing compounds are alkylene oxides and in particular loweralkylene oxides such as ethylene oxide, propylene oxide, butylene oxide,alcohol epoxides such as glycidol, and epihalohydrins such asepichlorohydrin, epibromohydrin, epiiodohydrin,1,2-epoxy-4-chlorobutane, 1,2-epoxy-4-bromobutane,1,2-epoxy-4-iodobutane, 2,3-epoxy-4-chlorobutane,2,3-epoxy-4-bromobutane, 2,3-epoxy-4-iodobutane,2,3-epoxy-5-chloropentane, 2,3-epoxy-5-bromopentane,1,2-epoxy-5-chloropentane, etc.; epoxy compounds such as2,2-bis(p-1,2-epoxypropoxyphenyl)-propane,1,4-bis(1,2-epoxypropoxy)benzene, N,N′-bis(2,3-epoxypropyl)piperazine,etc.

The terms “electrophilic group”, “electrophile” and the like as usedherein refers to an atom or group of atoms that can accept an electronpair to form a covalent bond. The “electrophilic group” used hereinincludes but is not limited to halide, carbonyl and epoxide containingcompounds. Common electrophiles may be halides such as thiophosgene,glycerin dichlorohydrin, phthaloyl chloride, succinyl chloride,chloroacetyl chloride, chlorosuccinyl chloride, etc.; ketones such aschloroacetone, bromoacetone, etc.; aldehydes such as glyoxal, etc.;isocyanates such as hexamethylene diisocyanate, tolylene diisocyanate,meta-xylylene diisocyanate, cyclohexylmethane-4,4-diisocyanate, etc andderivatives of these compounds.

The terms “nucleophilic group”, “nucleophile” and the like as usedherein refers to an atom or group of atoms that have an electron paircapable of forming a covalent bond. Groups of this type may be ionizablegroups that react as anionic groups. The “nucleophilic group” usedherein includes but is not limited to hydroxyl, primary amines,secondary amines, tertiary amines and thiols.

The term “ether” or “ether containing” refers to a class of organiccompounds of general formula R—O—R, wherein R is carbon. The term“ether” or “ether containing” as used herein is intended to excludethose compounds where R is not carbon, for example sialyl ethers,Si—O—Si.

The term “polyamine” refers to an organic compound having at least twopositively amino groups selected from the group comprising primary aminogroups, secondary amino groups and tertiary amino groups. Accordingly, apolyamine covers diamines, triamines and higher amines.

The term “biodegradable” or “biodegradable polymer” as used hereinrefers to environmentally-friendly materials that are degradable and/orcompostable. Such materials may be degradable/compostable by variousliving organisms or by exposure to light and/or oxygen. Therefore, theterm “biodegradable”, as used herein, will be understood to includematerials that are oxobiodegradable, photobiodegradable and microbiallybiodegradable.

The term “biocompatible” or “biocompatible polymer” refers to polymerswhich, in the amounts employed, are non-toxic, non-migratory, chemicallyinert, and substantially non-immunogenic when used in contact withbiological fluids, for example plasma or blood. Suitable biocompatiblepolymers include, by way of example, polysaccharides such as celluloseor chitin.

The term “biopolymer” refers to polymers that are produced by or derivedfrom living organisms. Exemplary biopolymers include polypeptides,nucleic acids and polysaccharides, for example cellulose and chitin.

The term “functional”, when used to describe a molecule or substance,refers to a group of atoms arranged in a way that determines thechemical properties of the substance and the molecule to which it isattached. Examples of functional groups include halogen atoms, amidegroups, hydroxyl groups, carboxylic acid groups and the like.

The term “target molecule” refers to a molecule that is to be detected,isolated, or tested for, and that is capable of reacting with or bindingto a functional substance such as a biological substance. Exemplarytarget molecules include proteins, polysaccharides, glycoproteins,hormones, receptors, lipids, small molecules, drugs, metabolites,cofactors, transition state analogues and toxins, or any nucleic acidthat is not complementary to its cognate nucleic acid. The targetmolecule may be in vivo, in vitro, in situ, or ex vivo.

The term “functional substances” and the like, used herein refersbroadly to mean molecules or active substances having a site capable ofreacting with or bonding with or having an affinity with a targetmolecule. The term “functional substances” and the like broadlyencompasses the biological substances and biomolecules.

The terms “biological substances” or “biomolecules” and the like, usedherein, refer to any substances and compounds substantially ofbiological origin. Hence, the terms encompass not only native molecules,such as those that can be isolated from natural sources, but also forms,fragments and derivatives derived therefrom, as well as recombinantforms and artificial molecules, as long as at least one property of thenative molecules is present. Hence, the term covers organic moleculesthat are produced by a living organism, including large polymericmolecules such as proteins, polysaccharides, and nucleic acids as wellas small molecules such as primary metabolites, secondary metabolites,and natural products.

The terms “biologically active substances”, “bioactive substances” andthe like, used herein, refer broadly to mean biological molecules orphysiologically active substances having a site capable of reacting withor bonding with or having an affinity with a target molecule. Thisincludes, but is not limited, to substances having a catalyticallyactive site such as enzymes, substances having a site capable of bondingto specific compounds or specific classes of compounds, such as nucleicacids oligonucleotides, deoxyribonucleic acid (DNA), ribonucleic acid(RNA), or lectins, vitamins, peptides, proteins, hormones, endocrinedisturbing chemicals, sugars, lipids and the like.

The term “poorly reactive substrate” means a substrate that is composedof a material that does not appreciably react chemically or biologicallywith a functional or biological substance as defined above. In someembodiments, the functional or biological substance may comprise abiomolecule and the non-reactive substrate is composed of a materialthat is bio-compatible in that the substrate material is not toxic anddoes not cause any adverse health effect to the human body. Non-reactivesubstrates that are also biocompatible are typically polymeric materialsthat are generally insoluble, flexible and which can conform to manydifferent shapes, including curved surfaces. It is noted that the term“polymer” is used to denote a chemical compound with high molecularweight consisting of a number of structural units linked together bycovalent bonds. One exemplary polymeric material that is non-reactiveand biocompatible with biological substances as defined above is thepolysaccharide cellulose.

The terms “linker” and “spacer” as used herein refer to an organicmoiety that connects two parts of a compound.

As used herein, the term “alkyl” includes within its meaning monovalent(“alkyl”) and divalent (“alkylene”) straight chain or branched chain orcyclic saturated aliphatic groups having from 1 to 25 carbon atoms, eg,1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24 or 25 carbon atoms. For example, the term alkyl includes,but is not limited to, methyl, ethyl, 1-propyl, isopropyl, 1-butyl,2-butyl, isobutyl, tert-butyl, amyl, 1,2-dimethylpropyl,1,1-dimethylpropyl, pentyl, isopentyl, hexyl, 4-methylpentyl,1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 2,2-dimethylbutyl,3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl,1,2,2-trimethylpropyl, 1,1,2-trimethylpropyl, 2-ethylpentyl,3-ethylpentyl, heptyl, 1-methylhexyl, 2,2-dimethylpentyl,3,3-dimethylpentyl, 4,4-dimethylpentyl, 1,2-dimethylpentyl,1,3-dimethylpentyl, 1,4-dimethylpentyl, 1,2,3-trimethylbutyl,1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 5-methylheptyl,1-methylheptyl, octyl, nonyl, decyl, and the like. Lower alkyls arealkyl groups as defined above 1 to 6 carbon atoms, preferably 1 to 4carbon atoms.

The term “alkenyl ” as used herein includes within its meaningmonovalent (“alkenyl”) and divalent (“alkenylene”) straight or branchedchain or cyclic unsaturated aliphatic hydrocarbon groups having from 2to 25 carbon atoms, eg, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 carbon atoms and having atleast one double bond, of either E, Z, cis or trans stereochemistrywhere applicable, anywhere in the alkyl chain. Examples of alkenylgroups include but are not limited to vinyl, allyl, 1-methylvinyl,1-propenyl, 2-methyl-1-propenyl, 2-methyl-1-propenyl, 1-butenyl,2-butenyl, 3-butentyl, 1,3-butadienyl, 1-pentenyl, 2-pententyl,3-pentenyl, 4-pentenyl, 1,3-pentadienyl, 2,4-pentadienyl,1,4-pentadienyl, 3-methyl-2-butenyl, 1-hexenyl, 2-hexenyl, 3-hexenyl,1,3-hexadienyl, 1,4-hexadienyl, 2-methylpentenyl, 1-heptenyl,2-heptentyl, 3-heptenyl, 1-octenyl, 1-nonenyl, 1-decenyl, and the like.Lower alkenyls are alkenyl groups as defined above with 2 to 6 carbonatoms, preferably 2 to 4 carbon atoms.

The term “alkynyl” as used herein includes within its meaning monovalent(“alkynyl”) and divalent (“alkynylene”) straight or branched chain orcyclic unsaturated aliphatic hydrocarbon groups having from 2 to 10carbon atoms and having at least one triple bond anywhere in the carbonchain. Examples of alkynyl groups include but are not limited toethynyl, 1-propynyl, 1-butynyl, 2-butynyl, 1-methyl-2-butynyl,3-methyl-1-butynyl, 1-pentynyl, 1-hexynyl, methylpentynyl, 1-heptynyl,2-heptynyl, 1-octynyl, 2-octynyl, 1-nonyl, 1-decynyl, and the like.Lower alkynylene are alkynylene groups as defined above with 2 to 6carbon atoms, preferably 2 to 4 carbon atoms.

The term “aryl” as used herein refers to a mono- or multiple-cycliccarbocyclic ring system having one or more aromatic rings including, butnot limited to, phenyl, naphthyl, tetrahydronaphthyl, indanyl, indenyland the like. Aryl groups (including bicyclic aryl groups) can beunsubstituted or substituted with one to five substituents or more(typically one to five substituent for monocyclic aryl and more thanfive substituents for bicyclic/oligocylic aryl) independently selectedfrom the group consisting of alkyl, alkenyl, alkynyl, haloalkyl, alkoxy,thioalkoxy, hydroxy, mercapto, amino, alkylamino, dialkylamino,acylamino, aminoacyl, alkoxycarbonyl, aryloxycarbonyl, azido, cyano,halo, nitro, carboxaldehyde, carboxy, carboxamide, carbamide, carbamate,sulfate, sulfonate, sulfinate, phosphate, phosphonate, phosphinate,phosphine, and protected hydroxy. In addition, substituted aryl groupsinclude tetrafluorophenyl and pentafluorophenyl.

The term “heteroaryl”, whether used alone or as part of another group,refers to a substituted or unsubstituted aromatic heterocyclic ringsystem (monocyclic or bicyclic). Heteroaryl groups can have, forexample, from about 3 to about 50 carbon atoms. Heteroaryl groupstypically include aromatic heterocyclic rings systems having about 4 toabout 14 ring atoms and containing carbon atoms and 1, 2, 3, or 4heteroatoms selected from oxygen, nitrogen or sulfur. Exemplaryheteroaryl groups include but are not limited to furan, thiophene,indole, azaindole, oxazole, triazole, isoxazole, isothiazole, imidazole,N-methylimidazole, pyridine, pyrimidine, pyrazine, pyrrole,N-methylpyrrole, pyrazole, N-methylpyrazole, 1,3,4-oxadiazole,1,2,4-triazole, 1-methyl-1,2,4-triazole, 1H-tetrazole,1-methyltetrazole, benzoxazole, benzothiazole, benzofuran,benzisoxazole, benzimidazole, N-methylbenzimidazole, azabenzimidazole,indazole, quinazoline, quinoline, and isoquinoline. Bicyclic aromaticheteroaryl groups include phenyl, pyridine, pyrimidine or pyridizinerings that are (a) fused to a 6-membered aromatic (unsaturated)heterocyclic ring having one nitrogen atom; (b) fused to a 5- or6-membered aromatic (unsaturated) heterocyclic ring having two nitrogenatoms; (c) fused to a 5-membered aromatic (unsaturated) heterocyclicring having one nitrogen atom together with either one oxygen or onesulfur atom; or (d) fused to a 5-membered aromatic (unsaturated)heterocyclic ring having one heteroatom selected from O, N or S. Theterm “heteroaryl” also includes aromatic heterocyclic rings that aresubstituted, for example with 1 to 5 substituents independently selectedfrom the group consisting of alkyl, alkenyl, alkynyl, haloalkyl, alkoxy,thioalkoxy, hydroxy, mercapto, amino, alkylamino, dialkylamino,acylamino, aminoacyl, alkoxycarbonyl, aryloxycarbonyl, azido, cyano,halo, nitro, carboxaldehyde, carboxy, carboxamide, carbamide, carbamate,sulfate, sulfonate, sulfinate, phosphate, phosphonate, phosphinate,phosphine, and protected hydroxy.

The term “optionally substituted” as used herein means the group towhich this term refers may be unsubstituted, or may be substituted withone or more groups independently selected from alkyl, alkenyl, alkynyl,aryl, heteroaryl, thioalkyl, cycloalkyl, cycloalkenyl, heterocycloalkyl,halo, carboxyl, carboxyalkyl, haloalkyl, haloalkynyl, hydroxy, alkoxy,thioalkoxy, mercapto, alkenyloxy, haloalkoxy, haloalkenyloxy, nitro,amino, nitroalkyl, nitroalkenyl, nitroalkynyl, nitroheterocyclyl,alkylamino, dialkylamino, alkenylamine, alkynylamino, acyl, alkenoyl,alkynoyl, acylamino, diacylamino, aminoacyl, acyloxy, alkylsulfonyloxy,heterocycloxy, heterocycloamino, haloheterocycloalkyl, alkoxycarbonyl,aryloxycarbonyl, azido, carboxaldehyde, carboxy, carboxamide, carbamide,carbamate, oxime, hydroxylamine, sulfate, sulfonate, sulfinate,alkylsulfenyl, alkylcarbonyloxy, alkylthio, acylthio,phosphorus-containing groups such as phosphate, phosphonate, phosphinateand phosphine, aryl, heteroaryl, alkylaryl, alkylheteroaryl, cyano,cyanate, isocyanate, —C(O)NH(alkyl), —C(O)N(alkyl)₂ and —C(O)NR′R″,where R′ and R″ are independently hydrogen, alkyl, aryl or heteroaryl asdefined herein.

The term “halogen” or variants such as “halide” or “halo” as used hereinrefers to fluorine, chlorine, bromine and iodine.

The term “amino” or “amine” as used herein refers to groups of the form—NR_(a)R_(b) wherein R_(a) and R_(b) are individually selected from thegroup including but not limited to hydrogen, optionally substitutedalkyl, optionally substituted alkenyl, optionally substituted alkynyl,and optionally substituted aryl groups.

The terms “chemically coupled” and “chemically couple” and grammaticalvariations thereof refer to the covalent and noncovalent bonding ofmolecules and include specifically, but not exclusively, covalentbonding, electrostatic bonding, hydrogen bonding and van der Waals'bonding. The terms encompass both indirect and direct bonding ofmolecules. Thus, if a first compound is chemically coupled to a secondcompound, that connection may be through a direct chemical bond, orthrough an indirect chemical bond via other compounds, linkers orconnections.

As used herein, the term “urease unit”, or urease “enzymatic unit”, [U],refers to that amount of enzyme (urease), which causes the liberation ofone micromole of ammonia per minute at 23° C. and pH 7.5.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

Unless specified otherwise, the terms “comprising” and “comprise”, andgrammatical variants thereof, are intended to represent “open” or“inclusive” language such that they include recited elements but alsopermit inclusion of additional, unrecited elements.

As used herein, the term “about”, in the context of concentrations ofcomponents of the formulations, typically means +/−5% of the statedvalue, more typically +/−4% of the stated value, more typically +/−30 ofthe stated value, more typically, +/−2% of the stated value, even moretypically +/−1% of the stated value, and even more typically +/−0.5% ofthe stated value.

Throughout this disclosure, certain embodiments may be disclosed in arange format. It should be understood that the description in rangeformat is merely for convenience and brevity and should not be construedas an inflexible limitation on the scope of the disclosed ranges.Accordingly, the description of a range should be considered to havespecifically disclosed all the possible sub-ranges as well as individualnumerical values within that range. For example, description of a rangesuch as from 1 to 6 should be considered to have specifically disclosedsub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4,from 2 to 6, from 3 to 6 etc., as well as individual numbers within thatrange, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of thebreadth of the range.

DETAILED DISCLOSURE OF EMBODIMENTS

Exemplary, non-limiting embodiments of a method of preparing a substratefor immobilization of functional substances thereon and a substrate forimmobilization of functional molecules thereon, will now be disclosed.

The substrate has compounds disposed thereon for immobilizing afunctional molecule, each compound having a chain comprising: a moiety Rthat is chemically coupled to the substrate, said moiety R beingselected from the group consisting of an ether, ester, carbonyl,carbonate ester, thioether, disulfide, sulfinyl, sulfonyl, andcarbonothioyl; and an epoxide-containing moiety that is coupled to themoiety R by a linker comprising at least one nucleophilic group.

In one embodiment, the moiety R is further selected from the groupconsisting of an amine, amide, carbamide, ureas and guanidines.

In one embodiment, the nucleophilic group excludes at least one ofoxygen-containing moieties and sulfur-containing moieties.

In another embodiment, the substrate comprises an additional epoxidecontaining group coupled to the chain. In one embodiment, the number ofadditional epoxide containing group is selected from the number 1, 2, 3,4 and 5. In another embodiment, at least one of the additional epoxidecontaining groups is coupled to said chain by the nucleophilic group ofsaid linker.

The linker may comprise additional nucleophilic groups to which saidadditional epoxide containing groups are coupled to said chain. Inanother embodiment, the additional epoxide-containing groups may branchfrom the chain by coupling with the additional nucleophilic groups ofsaid linker.

In one embodiment, the nucleophilic group of said linker is an amine.The linker may be selected from the group consisting of saturated andunsaturated aliphatic and aromatic amines, diamines, and triamines. Inone embodiment, the aliphatic groups of said amines are alkyl groups.

In another embodiment, the linker may contain an epoxide group.

In another embodiment, the linker comprises a di-nucleophilic species.The di-nucleophilic linker may be selected from at least one of analkyl-diamine and an alkene-diamine. In one embodiment, thedi-nucleophilic linker is selected from at least one of ethane-diamine,propane-diamine, butane-diamine, pentane-diamine, hexane-diamine. In oneembodiment, the di-nucleophilic linker is hexane-diamine.

In another embodiment, the epoxide containing-compound is derived byreaction of an epihalohydrin with the nucleophilic groups of saidlinker.

In one embodiment, the substrate may be inert to a functional moleculebeing immobilized by said epoxide-containing group.

In another embodiment, the substrate may be a polymer. The polymer maybe a biocompatible polymer. In another embodiment, the biocompatiblepolymer may be selected from the group consisting of a polyestersubstrate, a polyamide substrate, a polyacrylate substrate, and apolysaccharide-based substrate. In one embodiment, the polymer is apolysaccharide-based substrate which may be selected from the groupconsisting of cotton linters, cotton pulp, cotton fabrics, cellulosefibers, cellulose beads, cellulose powder, microcrystalline cellulose,cellulose membranes, rayon, cellophane, cellulose acetate, celluloseacetate membranes, chitosan, chitin, dextran derivatives and agarosederivatives.

In another embodiment, the polymer is a biopolymer. The biopolymer maybe selected from cellulose, chitosan, chitin, dextran, agarose andderivatives thereof.

In another embodiment, the substrate may comprise a coating disposed onsaid substrate, the coating comprising a substantially homogenousmixture of stabilizing additives selected to stabilize said functionalmolecule. In one embodiment, the stabilizing additives may be selectedfrom the group consisting of a sugar, an organic acid, an amino acid, asugar acid and a thiol.

In another embodiment, there is provided a method of immobilizing afunctional molecule on a substrate. The method comprises the step ofexposing the functional molecule to the substrate as described herein.

In one embodiment the functional molecule is selected from a groupconsisting of an affinity ligand, a chelator, a catalyst, an ionexchanger, a dye, an indicator and a biomolecule. In another embodiment,the functional molecule is chiral. In another embodiment, the functionalmolecule is a biomolecule. The biomolecule may be an enzyme. The enzymemay be selected from the group consisting of urease, uricase,creatininase, lipases, esterases, cellulases, amylases, pectinases,catalases, acylase, catalase, esterase, penicillin amidase,proteinase-K.

In another embodiment, the method further comprises the step of applyinga substantially homogenous mixture of stabilizing additives to thesurface of the substrate to stabilize selected to stabilize saidfunctional molecule. The step of applying the substantially homogenousmixture of additives comprises evaporating the solvent of a solution ofsaid additives onto the substrate. In one embodiment, the stabilizingadditives are selected from the group consisting of a sugar, an organicacid, an amino acid, a sugar acid and a thiol.

In another embodiment, there is also provided a method of preparing asubstrate for immobilization of functional molecules thereon. The methodcomprises the steps of: (i) providing electrophilic compounds coupled tothe surface of the substrate; (ii) allowing the electrophilic compoundsto undergo a nucleophilic substitution reaction to provide anucleophilic group thereon and thereby increase the nucleophilicity ofthe substrate surface; (iii) allowing the nucleophilic group to undergoa nucleophilic substitution reaction with another electrophilic compoundto provide an electrophilic group on the substrate surface and therebyincrease the electrophilicity of the substrate.

In one embodiment, the steps (ii) and (iii) may be repeated n number oftimes to form n generations of electrophilic groups on said substrate.In one embodiment, steps (ii) and (iii) are repeated 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 or more times.

In one embodiment, said step of providing electrophilic compoundscoupled to the surface of the substrate comprises chemically coupling afirst electrophilic compound to the substrate.

In another embodiment, step (ii) comprises the step of reacting anucleophile with the first electrophilic compound. In anotherembodiment, step (iii) comprises chemically coupling a secondelectrophilic compound to the nucleophile.

The electrophilic compound may be an epoxide-containing compound. In oneembodiment, the electrophilic compound may be selected from epoxycompounds such as alkylene oxides, alcohol epoxides, and epihalohydrins,halides The electrophilic compound may also include ketones, aldehydes,isocyanates and derivatives of these compounds.

In another embodiment, the epoxide-containing compound is anepihalohydrin. In one embodiment, the epihalohydrin may be selected fromthe group consisting of epichlorohydrin, epibromohydrin, epiiodohydrin,1,2-epoxy-4-chlorobutane, 1,2-epoxy-4-bromobutane,1,2-epoxy-4-iodobutane, 2,3-epoxy-4-chlorobutane,2,3-epoxy-4-bromobutane, 2,3-epoxy-4-iodobutane,2,3-epoxy-5-chloropentane, 2,3-epoxy-5-bromopentane,1,2-epoxy-5-chloropentane. In one embodiment, the epihalohydrin isepichlorohydrin.

In one embodiment, the nucleophile is a di-nucleophile or apolynucleophile. In another embodiment, the nucleophile comprises anamine. The amine may be selected from the group consisting of saturatedand unsaturated aliphatic or aromatic amines, diamines, triamines andhigher polyamines. In one embodiment, the aliphatic group of said aminesis selected from an alkyl group. In one embodiment, the amine may beselected from at least one of ethane-diamine, proane-diamine,butane-diamine, pentane-diamine, hexane-diamine. In one embodiment, theamine is hexane-diamine.

The substrate may comprise a polymer. The polymer may be a biocompatiblepolymer. In one embodiment, the biocompatible polymer may be selectedfrom the group consisting of a polyester substrate, a polyamidesubstrate, a polyacrylate substrate, and a polysaccharide-basedsubstrate.

In one embodiment, the substrate is a polysaccharide-based substrate.The polysaccharide-based substrate may selected from the groupconsisting of cotton linters, cotton pulp, cotton fabrics, cellulosefibers, cellulose beads, cellulose powder, microcrystalline cellulose,cellulose membranes, rayon, cellophane, cellulose acetate, celluloseacetate membranes, chitosan, chitin, dextran derivatives and agarosederivatives.

In another embodiment, the polymer may be a biopolymer. The biopolymermay be selected from cellulose, chitosan, chitin, dextran, agarose andderivatives thereof.

In another embodiment, the functional molecule may be selected from thegroup consisting of an affinity ligand, a chelator, a catalyst, an ionexchanger, a dye, an indicator and a biomolecule. In one embodiment, thefunctional molecule may be chiral. In another embodiment the functionalmolecule is a biomolecule. The biomolecule may be an enzyme selectedfrom the group consisting of urease, uricase, creatininase, lipases,esterases, cellulases, amylases, pectinases, catalases, acylase,catalase, esterase, penicillin amidase, proteinase-K.

In another embodiment, the method may further comprise the step ofapplying a substantially homogenous mixture of stabilizing additives tothe surface of the substrate wherein said stabilizing additives areselected to stabilize said functional molecule. The step of applying thesubstantially homogenous mixture of additives may comprise evaporatingthe solvent of a solution of said additives onto the substrate. In oneembodiment the stabilizing additives may be selected from the groupconsisting of a sugar, an organic acid, an amino acid, a sugar acid anda thiol.

There is also provided a sorbent cartridge for use in a dialysis device,the sorbent cartridge comprising a substrate as described herein forimmobilizing urease.

There is also provided a dialyzer for use in a dialysis device, thedialyzer comprising a substrate as described herein for immobilizingurease.

There is also provided a dialysis method comprising the steps of:exposing a dialysate containing urea to a substrate as described herein;and removing the dialysate from said substrate.

There is also provided the use of the substrate as described herein in adialysis device.

In another embodiment there is provided the use of the substrate inaccordance with the disclosure as a solid phase material forchromatography (including chiral chromatography and affinitychromatography). In another embodiment, the disclosure provides the useof the substrate in sensors and biosensors.

In another embodiment there is provided a method of preparing asubstrate for immobilization of functional substances thereon, themethod comprising the steps of chemically coupling a first electrophiliccompound to the substrate; and chemically coupling a secondelectrophilic compound to the first electrophilic compound that has beencoupled to the substrate, wherein said second electrophilic compound,when coupled to said first electrophilic compound, is configured toimmobilize the functional substance thereon. In one embodiment, thefirst electrophilic compound is a di-electrophile and is chemicallybonded to the substrate due to a nucleophilic substitution reactionbetween one electrophilic group of the di-electrophile and anucleophilic group on the substrate.

As a result of this first reaction, a poorly reactive (nucleophilic)substrate is converted into a strongly reactive (electrophilic)substrate. The di-electrophilic reagent may be an epihalohydrin. It mayalso be one of the group comprising cyanogen bromide, bromoacetic acid,glutaric aldehyde, and the like. The second electrophilic compound maybe chemically directly bonded to the first electrophilic compound suchas via a chemical link. The second electrophilic compound may also beindirectly chemically bonded to the first electrophilic compound, forexample via a linker. In one embodiment, the first and secondelectrophilic compounds are monomers.

Prior to the step of chemically coupling a first electrophilic compoundto the substrate, the method may include the step of functionalizing thesubstrate such that the substrate comprises functional groups that arecapable of being chemically coupled to the first electrophilic compound.

In one embodiment, the method comprises the step of using a linker tocouple the second electrophilic compound to the first electrophiliccompound. The linker may also be neutrally charged. In one embodiment,the linker may also comprise an aliphatic C₁₋₂₅ chain that is saturatedor unsaturated, straight or branched, which is optionally substituted,and wherein the carbons of the chain can be optionally replaced by—C(O)—, —C(O)C(O)—, —C(O)NR*—, —C(O)NR*NR*—, —CO₂—, —OC(O)—, —NR*CO₂—,—O—, —NR*C(O)NR*—, —OC(O)NR*—, —NR*NR*—, —NR*C(O)—, —S—, —SO—, —SO₂—,—NR*—, —SO₂NR*—, —NR*SO₂—, —C(O)NRO— or —NRC(NR)NR—, wherein R* isselected from hydrogen or C₁₋₁₀ aliphatic; wherein C₁₋₁₀ aliphatic canbe substituted or unsubstituted.

In one embodiment, the linker does not contain an epoxide group. Thelinker may also comprise at least one nucleophilic group. The linker maybe a multi-nucleophilic linker, that is, the linker may contain morethan one nucleophilic group. In one embodiment, the linker is adi-nucleophilic linker. When the linker is a di-nucleophilic linker, atleast one of the nucleophiles of the di-nucleophilic linker may beselected from the group consisting of NH, NR, NHO, NRO, O, S, Se, COO,CONH, CONR, CSS, COS, CONHO, CONRO, CONHNH, CONRNH, CONR¹NR², CNO, PHand PR,

where R, R¹ and R² are independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl.

When the functional linker does not contain an epoxide group and is adi-nucleophilic linker, the linker may have a general formula (I):

wherein:

X and Y are independently selected from NH, NR, O, S, COO, CONH andCONR;

R is selected from the group consisting of hydrogen, optionallysubstituted alkyl, optionally substituted alkenyl, optionallysubstituted alkynyl, optionally substituted aryl and optionallysubstituted heteroaryl; and

n is an integer from 0 to 25.

In another embodiment, the di-nucleophilic linker has the generalformula (II):

wherein:

X and Y are independently selected from NH, NR, NHO, NRO, O, S, Se, COO,CONH, CONR, CSS, COS, CONHO, CONRO CONHNH, CONRNH, CONRNR, CNO, PH, PR;

R, R¹, R², R³, R⁴ are independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl; and

m, n, p and q is an integer independently selected from 0 to 25.

The position of the groups

of formula (II) may be interchanged and these groups may also be presentin more than one positions as will be understood by a skilled artisan.

In another embodiment, the di-nucleophilic linker has the generalformula (IIa):

wherein:

X and Y are independently selected from the group consisting of NR¹R²,NRO, OR, SR, SeR, COOR, CONR, CSSR, COSR, CONRO, CONRNR¹R², CNOR andPR¹R², and any other substituents which may form cationic adducts;

R, R¹ and R² are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl; and

n is an integer from 0 to 25.

In one embodiment, the variables X and Y may also be any nucleophilicgroup that is capable of reacting with an epoxide group to form achemical bond.

The dinucleophilic linker may comprise an alky-diamine group. In oneembodiment, the di-nucleophilic linker is at least one ofethylene-diamine and hexanediamine. In another embodiment, the linkermay be a charged compound comprising nucleophiles such as NR¹R² where,R¹ and R² are defined above. The linker may also be small compoundsselected from the group consisting of H₂O, H₂S, H₂Se, PH₃, PH₂R, NH₃,NH₂R and NHR¹R², where, R, R¹ and R² are as defined above.

The linker may or may not be an epoxide-containing compound. In oneembodiment, when the linker is an epoxide containing compound, thelinker may have a general formula (Ia):

wherein:

X is selected from NH, NR, O, S, Se, COO, CONR¹NR², CONRO, CONH andCONR; R¹ and R² are independently selected from the group consisting ofhydrogen, optionally substituted alkyl, optionally substituted alkenyl,optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl; and

n is an integer from 0 to 25.

In another embodiment, the epoxide-containing linker has the generalformula (Ib):

wherein:

X is selected from NH, NR, NHO, NRO, O, S, Se, COO, CONH, CONR, CSS,COS, CONHO, CONRO CONHNH, CONRNH, CONRNR, CNO, PH, PR;

R, R¹, R², R³, R⁴ are independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkeny, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl; and

m, n, p and q is an integer independently selected from 0 to 25.

The position of the groups

of formula (II) may be interchanged and these groups may also be presentin more than one positions as will be understood by a skilled artisan.

In one embodiment, the variable X may also be any nucleophilic groupthat is capable of reacting with an epoxide group to form a chemicalbond.

The epoxy-containing linker may comprise hydroxy-oxiranes. In oneembodiment, the epoxy-containing linker is glycidol.

The disclosed method may also further comprise the steps of chemicallycoupling a subsequent electrophilic compound or ambiphilic compound tothe preceding electrophilic compound directly or indirectly via thefunctional linker disclosed above. These additional steps of chemicallycoupling subsequent electrophilic compounds may be carried outrepeatedly until the desired chain length is achieved. Advantageously,by repeating these steps, the number of electrophilic sites such asactive oxirane sites for binding with the biological substances mayincrease, thereby increasing the probability and affinity of thebiological substance to the substrate. In one embodiment, when thelinker is an ambiphilic compound, the linker comprises glycidol.

In one embodiment, the electrophilic compounds disclosed herein compriseepoxide containing compounds. For example, the first electrophiliccompound and second electrophilic compound may be a first epoxidecontaining compound and a second epoxide containing compound. In oneembodiment, at least one of the first epoxide containing-compound andthe second epoxide containing-compound is an epihalohydrin. Theepihalohydrin may be selected from the group consisting of,epichlorohydrin, epibromohydrin and epiiodohydrin. In one embodiment,the method comprises selecting a poorly reactive substrate. Thesubstrate may be selected from the group consisting of a polyestersubstrate, a polyamide substrate, an epoxy resin substrate, apolyacrylate substrate, a hydroxyl-functionalized substrate and apolysaccharide-based substrate. In one embodiment, thepolysaccharide-based substrate is selected from the group consisting ofcotton linters, cotton pulp, cotton fabrics, cellulose fibers, cellulosebeads, cellulose powder, microcrystalline cellulose, cellulosemembranes, rayon, cellophane, cellulose acetate, cellulose acetatemembranes, chitosan, chitin, dextran derivatives and agarosederivatives.

In one embodiment, the chemically coupling steps are undertaken at atemperature range of from about −30° C. to about 100° C., from about 0°C. to about 70° C., from about 4° C. to about 30° C. or from about 10°C. to about 27° C., from about 40° C. to about 70° C., from about 23° C.to about 35° C. and from about 23° C. to about 30° C.

The functional substances may be biologically active and may comprisebiological substances and/or biomolecules. In one embodiment, thebiological substances are enzymes. The method may comprise the step ofchemically coupling an enzyme to said second electrophilic compound thathas been coupled to the first electrophilic compound. The step ofchemically coupling an enzyme to said second electrophilic compound mayinclude providing stabilizing and activating additives such as sugars,thiols, antioxidants and chelators.

The enzyme may be selected from the group consisting of oxidoreductases,transferases, hydrolases, lyases, isomerases and ligases.Oxidoreductases catalyze oxidation-reduction reactions, and thesubstrate oxidized is regarded as hydrogen or electron donor.Transferases catalyze transfer of functional groups from one molecule toanother. Hydrolases catalyze hydrolytic cleavage of various bonds.Lyases catalyze cleavage of various bonds by other means than byhydrolysis or oxidation, meaning for example that they catalyze removalof a group from or addition of a group to a double bond, or othercleavages involving electron rearrangement. Isomerases catalyzeintramolecular rearrangement, meaning changes within one molecule.Ligases catalyze reactions in which two molecules are joined.

In one embodiment, the enzymes are oxidoreductases, which may act ondifferent groups of donors, such as the CH—OH group, the aldehyde or oxogroup, the CH—CH group, the CH—NH₂ group, the CH—NH group, NADH orNADPH, nitrogenous compounds, a sulfur group, a heme group, diphenolsand related substances, hydrogen, single donors with incorporation ofmolecular oxygen, paired donors with incorporation or reduction ofmolecular oxygen or others. Oxidoreductases may also be acting on CH₂groups or X—H and Y—H to form an X—Y bond. Typically enzymes belongingto the group of oxidoreductases may be referred to as oxidases,oxygenases, hydrogenases, dehydrogenases, reductases or the like.Exemplary oxidoreductases includes oxidases such as malate oxidase,glucose oxidase, hexose oxidase, aryl-alcohol oxidase, alcohol oxidase,long-chain alcohol oxidase, glycerol-3-phosphate oxidase, polyvinyl-alcohol oxidase, D-arabinono-1,4-lactone oxidase, D-mannitoloxidase, xylitol oxidase, oxalate oxidase, carbon-monoxide oxidase,4-hydroxyphenylpyruvate oxidase, dihydrouracil oxidase, ethanolamineoxidase, L-aspartate oxidase, sarcosine oxidase, urate oxidase,methanethiol oxidase, 3-hydroxyanthranilate oxidase, laccase, catalase,fatty-acid peroxidase, peroxidase, diarylpropane peroxidase,ferroxidase, pteridine oxidase, columbamine oxidase and the like.Oxidoreductases may also include oxygenases such as catechol1,2-dioxygenase, gentisate 1,2-dioxygenase, homogentisate1,2-dioxygenase, lipoxygenase, ascorbate 2,3-dioxygenase,3-carboxyethylcatechol 2,3-dioxygenase, indole 2,3-dioxygenase, caffeate3,4-dioxygenase, arachidonate 5-lipoxygenase, biphenyl-2,3-diol1,2-dioxygenase, linoleate 11-lipoxygenase, acetylacetone-cleavingenzyme, lactate 2-monooxygenase, phenylalanine 2-monooxygenase, inositoloxygenase and the like. Oxidoreductases may also include dehydrogenasessuch as alcohol dehydrogenase, glycerol dehydrogenase,propanediol-phosphate dehydrogenase, L-lactate dehydrogenase, D-lactatedehydrogenase, glycerate dehydrogenase, glucose 1-dehydrogenase,galactose 1-dehydrogenase, allyl-alcohol dehydrogenase,4-hydroxybutyrate dehydrogenase, octanol dehydrogenase, aryl-alcoholdehydrogenase, cyclopentanol dehydrogenase, long-chain-3-hydroxyacyl-CoAdehydrogenase, L-lactate dehydrogenase, D-lactate dehydrogenase, butanaldehydrogenase, terephthalate 1,2-cis-dihydrodiol dehydrogenase,succinate dehydrogenase, glutamate dehydrogenase, glycine dehydrogenase,hydrogen dehydrogenase, 4-cresol dehydrogenase, phosphonatedehydrogenase and the like. Reductases belonging to the group ofoxidoreductases may also include enzymes such as diethyl2-methyl-3-oxosuccinate reductase, tropinone reductase,long-chain-fatty-acyl-CoA reductase, carboxylate reductase, D-prolinereductase, glycine reductase, Heme-proteins such as cytochromes and thelike. In one embodiment, the enzymes are lyases, which may belong toeither of the following groups: carbon-carbon lyases, carbon-oxygenlyases, carbon-nitrogen lyases, carbon-sulfur lyases, carbon-halidelyases, phosphorus-oxygen lyases and other lyases.

The carbon-carbon lyases may also include carboxy-lyases,aldehyde-lyases, oxo-acid-lyases and others. Some specific examplesbelonging to these groups are oxalate decarboxylase, acetolactatedecarboxylase, aspartate 4-decarboxylase, lysine decarboxylase,aromatic-L-amino-acid decarboxylase, methylmalonyl-CoA decarboxylase,carnitine decarboxylase, indole-3-glycerol-phosphate synthase, gallatedecarboxylase, branched-chain-2-oxoacid, decarboxylase, tartratedecarboxylase, arylmalonate decarboxylase, fructose-bisphosphatealdolase, 2-dehydro-3-deoxy-phosphogluconate aldolase,trimethylamine-oxide aldolase, propioin synthase, lactate aldolase,vanillin synthase, isocitrate lyase, hydroxymethylglutaryl-CoA lyase,3-hydroxyaspartate aldolase, tryptophanase, deoxyribodipyrimidinephoto-lyase, octadecanal decarbonylase and the like.

The carbon-oxygen lyases may include hydro-lyases, lyases acting onpolysaccharides, phosphates and others. Some specific examples arecarbonate dehydratase, fumarate hydratase, aconitate hydratase, citratedehydratase, arabinonate dehydratase, galactonate dehydratase, altronatedehydratase, mannonate dehydratase, dihydroxy-acid dehydratase,3-dehydroquinate dehydratase, propanediol dehydratase, glyceroldehydratase, maleate hydratase, oleate hydratase, pectate lyase,poly(β-D-mannuronate)lyase, oligogalacturonide lyase,poly(α-L-guluronate)lyase, xanthan lyase, ethanolamine-phosphatephospho-lyase, carboxymethyloxysuccinate lyase and the like.

The carbon-nitrogen lyases may include ammonia-lyases, lyases acting onamides, amidines, etc., amine-lyases and the like. Specific examples ofthese groups of lyases are aspartate ammonia-lyase, phenylalanineammonia-lyase, ethanolamine ammonia-lyase, glucosaminate ammonia-lyase,argininosuccinate lyase, adenylosuccinate lyase, ureidoglycolate lyaseand 3-ketovalidoxylamine C—N-lyase.

The carbon-sulfur lyases may include dimethylpropiothetindethiomethylase, alliin lyase, lactoylglutathione lyase and cysteinelyase.

The carbon-halide lyases may include 3-chloro-D-alaninedehydrochlorinase and dichloromethane dehalogenase.

The phosphorus-oxygen lyases may include adenylate cyclase, cytidylatecyclase, glycosylphosphatidylinositol diacylglycerol-lyase.

In another embodiment, the enzymes are hydrolases selected from thegroup consisting of glycosylases, enzymes acting on acid anhydrides andenzymes acting on specific bonds such as ester bonds, ether bonds,carbon-nitrogen bonds, peptide bonds, carbon-carbon bonds, halide bonds,phosphorus-nitrogen bonds, sulfur-nitrogen bonds, carbon-phosphorusbonds, sulfur-sulfur bonds or carbon sulfur bonds.

The glycosylases may be glycosidases, which are capable of hydrolysingO- and S-glycosyl compounds or N-glycosyl compounds. The glycosylasesmay also include α-amylase, β-amylase, glucan 1,4-α-glucosidase,cellulase, endo-1,3(4)-β-glucanase, inulinase, endo-1,4-β-xylanase,oligo-1,6-glucosidase, dextranase, chitinase, pectinase,polygalacturonase, lysozyme, levanase, quercitrinase, galacturan1,4-α-galacturonidase, isoamylase, glucan 1,6-αglucosidase, glucanendo-1,2-α-glucosidase, licheninase, agarase,exo-poly-α-galacturonosidase, κ-carrageenase, steryl-β-glucosidase,strictosidine β-glucosidase, mannosyl-oligosaccharide glucosidase,lactase, oligo-xyloglucan β-glycosidase, polymannuronate hydrolase,chitosanase, poly(ADP-ribose) glycohydrolase, purine nucleosidase,inosine nucleosidase, uridine nucleosidase, adenosine nucleosidase, andthe like.

The enzymes acting on acid anhydrides may be for example those acting onphosphorus- or sulfonyl-containing anhydrides. Exemplary enzymes actingon acid anhydrides are include inorganic diphosphatase,trimetaphosphatase, adenosine-triphosphatase, apyrase,nucleoside-diphosphatase, acylphosphatase, nucleotide diphosphatase,endopolyphosphatase, exopolyphosphatase, nucleosidephospho-acylhydrolase, triphosphatase, CDP-diacylglyceroldiphosphatase,undecaprenyldiphosphatase, dolichyldiphosphatase,oligosaccharide-diphosphodolichol diphosphatase, heterotrimericG-protein GTPase, small monomeric GTPase, dynamin GTPase, tubulinGTPase, diphosphoinositolpolyphosphate diphosphatase, H⁺-exportingATPase, monosaccharide-transporting ATPase, maltose-transporting ATPase,glycerol-3-phosphate-transporting ATPase, oligopeptide-transportingATPase, polyamine-transporting ATPase, peptide-transporting ATPase,fatty-acyl-CoA-transporting ATPase, protein-secreting ATPase and thelike.

The enzymes acting on the ester bonds may include esterases, lipases,carboxylic ester hydrolases, thiolester hydrolases, phosphoric esterhydrolases, sulfuric ester hydrolases and ribonucleases. Exemplaryenzymes acting on ester bonds include acetyl-CoA hydrolase,palmitoyl-CoA hydrolase, succinyl-CoA hydrolase, 3-hydroxyisobutyryl-CoAhydrolase, hydroxy-methylglutaryl-CoA hydrolase, hydroxyacylglutathionehydrolase, glutathione thiolesterase, formyl-CoA hydrolase,acetoacetyl-CoA hydrolase, S-formylglutathione hydrolase,5-succinylglutathione hydrolase, oleoyl-[acyl-carrier-protein]hydrolase,ubiquitin thiolesterase, [citrate-(pro-35)-lyase]thiolesterase,(S)-methyl-malonyl-CoA hydrolase, ADP-dependent short-chain-acyl-CoAhydrolase, ADP-dependent medium-chain-acyl-CoA hydrolase, acyl-CoAhydrolase, dodecanoyl-[acyl-carrier protein]hydrolase,palmitoyl-(protein)hydrolase, 4-hydroxy-benzoyl-CoA thioesterase,2-(2-hydroxyphenyl)benzene-sulfinate hydrolase, alkaline phosphatase,acid phosphatase, phosphoserine phosphatase, phosphatidate phosphatase,5′-nucleotidase, 3′-nucleotidase, 3′(2′),5′-bisphosphate nucleotidase,3-phytase, glucose-6-phosphatase, glycerol-2-phosphatase,phosphoglycerate phosphatase, glycerol-1-phosphatase,mannitol-1-phosphatase, sugar-phosphatase, sucrose-phosphatase,inositol-1 (or 4)-monophosphatase, 4-phytase,phosphatidylglycero-phosphatase, ADP phosphoglycerate phosphatase,N-acyl-neuraminate-9-phosphatase, nucleotidase, polynucleotide3′-phosphatase, glycogen-synthase-D phosphatase, pyruvatedehydrogenase(lipoamide)phosphatase, acetyl-CoA carboxylase phosphatase,3-deoxy-manno-octulosonate-8-phosphatase, polynucleotide 5′-phosphatase,sugar-terminal-phosphatase, alkylacetylglycerophosphatase,2-deoxyglucose-6-phosphatase, glucosylglycerol 3-phosphatase, 5-phytase,phosphodiesterase I, glycerophosphocholine phosphodiesterase,phospholipase C, phospholipase D, phosphoinositide phospholipase C,sphingomyelin phosphodiesterase, glycerophosphocholinecholinephosphodiesterase, alkylglycerophosphoethanolaminephosphodiesterase, glycerophosphoinositol glyce-rophosphodiesterase,arylsulfatase, steryl-sulfatase, glycosulfatase, choline-sulfatase,cellulose-polysulfatase, monomethyl-sulfatase, D-lactate-2-sulfatase,glucuronate-2-sulfatase, prenyl-diphosphatase, aryldialkylphosphatase,diisopropyl-fluorophosphatase, oligonucleotidase, poly(A)-specificribonuclease, yeast ribonuclease, deoxyribonuclease (pyrimidine dimer),Physarum polycephalum ribonuclease, ribonculease alpha, Aspergillusnuclease S1, Serratia marcescens nuclease, carboxylesterase,arylesterase, triacylglycerol lipase, phospholipase A2,lysophospholipase, acetylesterase, acetylcholinesterase, cholinesterase,tropinesterase, pectinesterase, sterol esterase, chlorophyllase,L-arabinonolactonase, gluconolactonase, uronolactonase, tannase,retinyl-palmitate esterase, hydroxybutyrate-dimer hydrolase,acylglycerol lipase, 3-oxoadipate enol-lactonase, 1,4-lactonase,galactolipase, 4-pyridoxolactonase, acylcarnitine hydrolase,aminoacyl-tRNA hydrolase, D-arabinono-lactonase,6-phosphogluconolactonase, phospholipase A1, 6-acetylglucosedeacetylase, lipoprotein lipase, dihydrocoumarin hydrolase,limonin-D-ring-lactonase, steroid-lactonase, triacetate-lactonase,actinomycin lactonase, orsellinate-depside hydrolase, cephalosporin-Cdeacetylase, chlorogenate hydrolase, α-amino-acid esterase,4-methyloxaloacetate esterase, carboxy-methylenebutenolidase,deoxylimonate-A-ring-lactonase, 1-alkyl-2-acetylglycerophosphocholineesterase, fusarinine-C-ornithinesterase, sinapine esterase, wax-esterhydrolase, phorbol-diester hydrolase, phosphatidylinositol deacylase,sialate O-acetylesterase, acetoxybutynyl-bithiophene deacetylase,acetylsalicylate deacetylase, methylumbelliferyl-acetate deacetylase,2-pyrone-4,6-dicarboxylate lactonase, N-acetylgalactosaminoglycandeacetylase, juvenile-hormone esterase, bis(2-ethyl-hexyl)phthalateesterase, protein-glutamate methyl-esterase, 11-cis-retinyl-palmitatehydrolase, all-trans-retinyl-palmitate hydrolase,L-rhamnono-1,4-lactonase, 5-(3,4-diacetoxybut-1-ynyl)-2,2′-bithiophenedeacetylase, fatty-acyl-ethyl-ester synthase, xylono-1,4-lactonase,cetraxate benzylesterase, acetylalkylglycerol acetylhydrolase,acetylxylan esterase, feruloyl esterase, cutinase,poly(3-hydroxybutyrate)depolymerase, poly(3-hydroxyoctanoate),depolymerase acyloxyacyl hydrolase, acyloxyacyl hydrolase,polyneuridine-aldehyde esterase and the like.

The enzymes acting on ether bonds may include trialkylsulfoniumhydrolases and ether hydrolases. Enzymes acting on ether bonds may acton both thioether bonds and on the oxygen equivalent. Specific enzymeexamples belonging to these groups are adenosylhomocysteinase,adenosylmethionine hydrolase, isochorismatase,alkenylglycerophosphocholine hydrolase, epoxide hydrolase,trarcs-epoxysuccinate hydrolase, alkenylglycerophosphoethanolaminehydrolase, leukotriene-A4 hydrolase, hepoxilin-epoxide hydrolase andlimonene-1,2-epoxide hydrolase.

The enzymes acting on carbon-nitrogen bonds may hydrolyze linear amides,cyclic amides, linear amidines, cyclic amidines, linear carbamides(ureas), cyclic carbamides (ureas), nitriles and other compounds.Specific examples belonging to these groups are urease, amidase(acylase), asparaginase, glutaminase, ω-amidase, β-ureidopropionase,arylformamidase, biotinidase, aryl-acylamidase, aminoacylase,aspartoacylase, acetyl-ornithine deacetylase, acyl-lysine deacylase,succinyl-diaminopimelate desuccinylase, pantothenase, ceramidase,choloylglycine hydrolase, N-acetylglucosamine-6-phosphate deacetylase,N-acetylmuramoyl-L-alanine amidase, 2-(acetamidomethylene)succinatehydrolase, 5-aminopentanamidase, formylmethionine deformylase, hippuratehydrolase, N-acetylglucosamine deacetylase, D-glutaminase,N-methyl-2-oxoglutaramate hydrolase, glutamin-(asparagin-)ase,alkylamidase, acylagmatine amidase, chitin deacetylase,peptidyl-glutaminase, N-carbamoyl-sarcosine amidase,N-(long-chain-acyl)ethanolamine deacylase, mimosinase, acetylputrescinedeacetylase, 4-acetamidobutyrate deacetylase, theanine hydrolase,2-(hydroxymethyl)-3-(acetamidomethylene)succinate hydrolase,4-methyleneglutaminase, N-formylglutamate deformylase, glycosphingolipiddeacylase, aculeacin-A deacylase, peptide deformylase,dihydropyrimidinase, dihydroorotase, carboxymethyl-hydantoinase,creatininase, L-lysine-lactamase, arginase, guanidinoacetase,creatinase, allantoicase, cytosine deaminase, riboflavinase, thiaminase,1-aminocyclopropane-1-carboxylate deaminase and the like.

In one embodiment, the enzymes immobilized are enzymes acting on peptidebonds, which group is also referred to as peptidases. Peptidases can befurther divided into exopeptidases that act only near a terminus of apolypeptide chain and endopeptidases that act internally in polypeptidechains. Enzymes acting on peptide bonds may include enzymes selectedfrom the group of aminopeptidases, dipeptidases, di- ortripeptidyl-peptidases, peptidyl-dipeptidases, serine-typecarboxypeptidases, metallocarboxypeptidases, cysteine-typecarboxypeptidases, omega peptidases, serine endopeptidases, cysteineendopeptidases, aspartic endopeptidases, metalloendopeptidases andthreonine endopeptidases. Some specific examples of enzymes belonging tothese groups are cystinyl aminopeptidase, tripeptide aminopeptidase,prolyl aminopeptidase, arginyl aminopeptidase, glutamyl aminopeptidase,cytosol alanyl aminopeptidase, lysyl aminopeptidase, Met-X dipeptidase,non-stereospecific dipeptidase, cytosol nonspecific dipeptidase,membrane dipeptidase, dipeptidase E, dipeptidyl-peptidase I,dipeptidyl-dipeptidase, tripeptidyl-peptidase I, tripeptidyl-peptidaseII, X-Pro dipeptidyl-peptidase, peptidyl-dipeptidase A, lysosomal Pro-Xcarboxypeptidase, carboxypeptidase C, acylaminoacyl-peptidase,peptidyl-glycinamidase, β-aspartyl-peptidase, ubiquitinyl hydrolase 1,chymo-trypsin, chymotrypsin C, metridin, trypsin, thrombin, plasmin,enteropeptidase, acrosin, α-Lytic endopeptidase, glutamyl endopeptidase,cathepsin G, cucumisin, prolyl oligopeptidase, brachyurin, plasmakallikrein, tissue kallikrein, pancreatic elastase, leukocyte elastase,chymase, cerevisin, hypodermin C, lysyl endopeptidase, endopeptidase La,γ-renin, venombin AB, leucyl endopeptidase, tryptase, scutelarin, kexin,subtilisin, oryzin, endopeptidase K, thermomycolin, thermitase,endopeptidase So, t-plasminogen activator, protein C (activated),pancreatic endopeptidase E, pancreatic elastase II, IgA-specific serineendopeptidase, u-plasminogen activator, venombin A, furin, myeloblasts,semenogelase, granzyme A, granzyme B, streptogrisin A, streptogrisin B,glutamyl endopeptidase II, oligopeptidase B, omptin, togavirin,flavivirin, endopeptidase Clp, proprotein convertase 1, proproteinconvertase 2, lactocepin, assemblin, hepacivirin, spermosin,pseudomonalisin, xanthomonalisin, C-terminal processing peptidase,physarolisin, cathepsin B, papain, ficain, chymopapain, asclepain,clostripain, streptopain, actinidain, cathepsin L, cathepsin H,cathepsin T, glycyl endopeptidase, cancer procoagulant, cathepsin S,picornain 3 C, picornain 2 A, caricain, ananain, stem bromelain, fruitbromelain, legumain, histolysain, caspase-1, gingipain R, cathepsin K,adenain, bleomycin hydrolase, cathepsin F, cathepsin O, cathepsin V,nuclear-inclusion-a endopeptidase, helper-component proteinase,proteinase K, L-peptidase, gingipain K, staphopain, separase, V-cathendopeptidase, cruzipain, calpain-1, calpain-2, pepsin A, pepsin B,gastricsin, chymosin, cathepsin D, nepenthesin, renin,Proopiomelanocortin converting enzyme, aspergillopepsin I,aspergillopepsin II, penicillopepsin, rhizopuspepsin, endothiapepsin,mucorpepsin, candidapepsin, saccharopepsin, rhodotorulapepsin,acrocylindropepsin, polyporopepsin, pycnoporopepsin, scytalidopepsin A,scytalidopepsin B, cathepsin E, barrierpepsin, signal peptidase II,plasmepsin I, plasmepsin II, phytepsin, yapsin 1, thermopsin, prepilinpeptidase, nodavirus endopeptidase, memapsin 1, memapsin 2, atrolysin A,microbial collagenase, leucolysin, stromelysin 1, meprin A, procollagenC-endopeptidase, astacin, pseudolysin, thermolysin, bacillolysin,aureolysin, coccolysin, mycolysin, gelatinase B, leishmanolysin,saccharolysin, gametolysin, serralysin, horrilysin, ruberlysin,bothropasin, oligopeptidase A, endothelin-converting enzyme, ADAM 10endopeptidase and the like.

The enzymes acting on carbon-carbon bonds may include, but are notlimited to oxaloacetase, fumarylacetoacetase, kynureninase, phloretinhydrolase, acylpyruvate hydrolase, acetylpyruvate hydrolase, β-diketonehydrolase, 2,6-dioxo-6-phenylhexa-3-enoate hydrolase,2-hydroxymuconate-semialdehyde hydrolase and cyclohexane-1,3-dionehydrolase.

The enzymes acting on halide bonds may include alkylhalidase, 2-haloaciddehalogenase, haloacetate dehalogenase, thyroxine deiodinase, haloalkanedehalogenase, 4-chlorobenzoate dehalogenase, 4-chlorobenzoyl-CoAdehalogenase, atrazine chlorohydrolase and the like.

The immobilized enzymes disclosed herein may also include enzymes actingon specific bonds such as phosphoamidase, N-sulfoglucosaminesulfohydrolase, cyclamate sulfohydrolase, phosphonoacetaldehydehydrolase, phosphonoacetate hydrolase, trithionate hydrolase, UDPsulfoquinovose synthase and the like.

Preferably, the enzymes are ureases. The enzyme may be chemicallycoupled to the second epoxide-containing compound that has been coupledto the first epoxide-containing compound. It may also be coupleddirectly to the first epoxy-containing compound.

The substrate obtained from the described method for immobilization ofbiological substances thereon has a ether-containing compound having onemoiety coupled to a substrate and another moiety coupled to anepoxide-containing compound. The substrate may be used in a dialysisdevice such as a peritoneal dialysis device or a hemodialysis device. Inone embodiment, the substrate is used in a sorbent of a hemodialysisdevice.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings illustrate a disclosed embodiment and serve toexplain the principles of the disclosed embodiment. It is to beunderstood, however, that the drawings are designed for purposes ofillustration only, and not as a definition of the limits of theinvention.

FIG. 1 a is a schematic of one embodiment of the disclosed method ofusing di-nucleophilic linkers.

FIG. 1 b is a schematic showing another possible modified substrate thatmay be obtained from the same embodiment of the method shown in FIG. 1a.

FIG. 2 is a schematic of another embodiment of the disclosed methodusing oxirane-functionalized linkers.

FIG. 3 is a schematic of a specific example of the method shown in FIG.1 a, when hexanediamine is used as a linker and epichlorohydrin is usedas the first and second epoxide containing compound.

FIG. 4 is a schematic of a specific example of the method shown in FIG.1 b, when glycidol is used as a linker.

DETAILED DESCRIPTION OF DRAWINGS

Referring to FIG. 1 a, there is shown a schematic of one embodiment ofthe disclosed method 100 of using di-nucleophilic linkers. A free(primary) hydroxyl group on the surface of an insoluble polymer 110 isfirst reacted with an epihalohydrin shown in step A-1. The reactionresults in the release of the halogen on the epihalohydrin and a protonon the hydroxyl group, forming an ether bond, such that the resulting,modified substrate 112 is now chemically coupled to an epoxide group atthe terminal end. The substrate 112 containing the epoxide group isthen, in step A-2, reacted with a di-nucleophilic linker having thegeneral formula (II), giving the linker modified substrate 114 as aproduct.

wherein:

X and Y are independently selected from NH, NR, NHO, NRO, O, S, Se, COO,CONH, CONR, CSS, COS, CONHO, CONRO CONHNH, CONRNH, CONRNR, CNO, PH, PR;

R, R¹, R², R³, R⁴ are independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl; and

m, n, p and q is an integer independently selected from 0 to 25.

After the reaction with the linker group in step A-2, the modifiedsubstrate 114 now contains the nucleophilic group Y at its terminal end.The nucleophilic group Y is then reacted in step A-3 with anotherepihalohydrin. Via nucleophilic substitution, the halogen present on theepihalohydrin is substituted by the nucleophilic group Y, resulting inthe modified substrate 116 now having an ether moiety 140, a terminalepoxy moiety 142, that are respectively coupled by the linker 144. Theepoxide terminal group of the modified substrate 116 is then reactedwith a biological substance in the form of enzyme 120 that contains anucleophilic group Z in step A-4. The enzyme becomes immobilized on thesubstrate to give the overall product 130. Stabilizers such as thiolsmay also be added in step A-4.

Referring to FIG. 1 b, there is shown a schematic 200 showing anotherproduct that may be obtained from the same embodiment of the methodshown in FIG. 1 a. The steps undertaken in FIG. 1 b are the same as FIG.1 a. However, in step B-3, more than one molecule of epihalohydrinundergoes nucleophilic substitution at both the nucleophilic groups Xand Y, resulting in a substrate having multiple epoxide groups.Accordingly, the final modified substrate obtained (216) differs frommodified substrate 116 in that the substrate 216 contains additionalepoxy moieties 242 at nucleophilic groups X and Y. Modified substrate216 can then undergo immobilization reactions similar to Step B-4 inFIG. 1 a.

Referring to FIG. 2, there is shown a schematic of one embodiment of thedisclosed method 300 using oxirane functionalized linkers. The substrate310 containing a hydroxy group is first reacted with an epihalohydrinshown in step C-1. The reaction results in the release of the halogen onthe epihalohydrin and hydrogen form the hydroxy group such that theresulting modified substrate 312 is now linked to an epoxide group atthe terminal end. The substrate 312 containing the epoxide group is thenin step C-2, reacted with at least one unit of an oxirane functionalizedlinker having the general formula (Ib):

wherein:

X is selected from NH, NR, NHO, NRO, O, S, Se, COO, CONH, CONR, CSS,COS, CONHO, CONRO CONHNH, CONRNH, CONRNR, CNO, PH, PR;

R, R¹, R², R³, R⁴ are independently selected from the group consistingof hydrogen, optionally substituted alkyl, optionally substitutedalkenyl, optionally substituted alkynyl, optionally substituted aryl andoptionally substituted heteroaryl; and

m, n, p and q is an integer independently selected from 0 to 25.

After the reaction with the linker in step C-2, the resulting modifiedsubstrate 314 now contains the epoxide group of the oxiranefunctionalized linker at its terminal end. The epoxide terminal group isthen reacted with a biological substance 320 that contains anucleophilic group Z in step C-3. Eventually the biological substance isimmobilized on the substrate to give the overall product 330.

Referring to FIG. 3 there is shown a schematic 400 of a specific exampleof the method shown in FIG. 1 a, but when hexanediamine is used as alinker in step D-2 and epichlorohydrin is used as the first and secondepoxide containing compound in steps D-1 and D-3. The resulting modifiedsubstrate contains one ether moiety 440 and at least one epoxy moiety442, as exemplified in substrate 416. It may also contain multiple epoxymoieties, as shown in modified substrates 450 and 452.

Referring to FIG. 4 there is shown a schematic 500 of a specific exampleof the method shown in FIG. 1 b, when glycidol is used as a linker instep E-2. The resulting product obtained is indicated by referencenumeral 514.

EXAMPLES

Non-limiting embodiments of the invention and a comparative example willbe further described in greater detail by reference to specificExamples, which should not be construed as in any way limiting the scopeof the invention.

Example 1 Preparation of Epoxy-Functionalized Substrate

Mercerization and epoxy-functionalization of cellulose was conducted bytreating a vigorously stirred suspension of 5.0 g of cellulose in 100 mlof 2.4M sodium hydroxide with 30 ml of epichlorohydrin at 55° C. for 4h. The reaction mixture was filtered by suction and the solid residue(“primary epoxy cellulose”) was washed with ultrapure water (3×50 ml).The epoxy group loading of dry primary epoxy cellulose was 125 μmol/g(see Table 1).

The primary epoxy-cellulose (15.3 g of wet material) was reacted with 15ml of hexane-diamine (70% aqueous solution) in 100 ml of methanol at 23°C. for 4 h. The reaction mixture was filtered by suction and washed oncewith 100 ml of methanol to give 9.8 g of wet “amino cellulose”. Thepresence of primary amino groups on the product was qualitativelyassessed by its reaction with ninhydrin.

The amino cellulose (9.6 g of wet material) was then reacted with 30 mlof epichlorohydrin in 100 ml of methanol at 23° C. for 4 h. The reactionproduct (“secondary epoxy cellulose”) was obtained by suction filtrationand washing with cold water (3×100 ml). The epoxy group loading of drysecondary epoxy cellulose product was 108 μmol/g (see Table 1).

Method for the Determination of the Epoxy Group Loading

A sample of the epoxy group containing material (about 1 g of wetmaterial) is suspended in 5 ml of water.

The suspension is titrated with 0.01N HCl to neutral pH if necessary.The neutral suspension is treated with 5 ml of 1.3M aqueous sodiumthiosulphate solution followed by incubation for 15 min with occasionalshaking. The suspension is then titrated with 0.01N HCl againstbromophenol blue. The total amount of epoxy groups present in the sampleis equivalent to the amount of HCl consumed in the titration. The epoxyloading of the dry material is calculated based on the known watercontents (LOD) of the wet material. Representative experimental valuesare summarized in Table 1.

Example 2 Immobilization of Urease

The secondary epoxy cellulose prepared in Example 1 (12.5 g of wetmaterial) was suspended in a cooled solution of Jack Bean urease (4.2 g)in 150 ml of 1.0M potassium phosphate buffer at pH 7.5. Theimmobilization reaction was carried out in an incubator shaker at 4° C.for 24 h.

The reaction mixture was then filtered by suction and the residue(“immobilized urease”) was washed 3 times with cold ultra pure water(3×150 ml).

Post-Immobilization Treatment of Immobilized Urease

The immobilized urease was soaked in an aqueous solution of cystein (5mg/ml), ethylenediaminetetraacetic acid (EDTA, 1.0 mM) and glucose (0.2g/ml) for 10 min, followed by suction filtration, and lyophilization for24 h.

COMPARATIVE EXAMPLE TABLES

TABLE 1 Comparison of epoxy group density of activated/modifiedcellulose and commercial Eupergit ® C (Sigma-Aldrich) Epoxy loadingEpoxy loading after step D-1 after step D-3 Eupergit (μmol/g) (μmol/g)(μmol/g) Titration 125 108 260 value Commercial N.A. N.A. ≧200 claimedvalue

TABLE 2 Comparison of the activity of immobilized urease onactivated/modified cellulose with and without amino linker Activatedcellulose Activated cellulose Substrate with amino linker without aminolinker Activity of 1100 <100 immobilized urease product (U/g)

TABLE 3 Comparison of activities of urease immobilized on a commercialsubstrate (Eupergit ® C) and on activated cellulose Activated SubstrateEupergit ® C cellulose Activity of 689 1850 immobilized urease product(U/g)

APPLICATIONS

The disclosed method of preparing a substrate is a cost effective andefficient way of producing a substrate that is capable of immobilizingfunctional substances thereon. Advantageously, the method ensures thatthe substrate produced by the method allows the functional substances,such as enzymes, to be stably immobilized thereon. More advantageously,as the enzymes are stably attached to the substrate, the substrate canbe reused repeatedly for long periods of time without substantiallylosing its enzymatic activity.

As the disclosed method can also work with low cost starting materials,the overall production costs can be substantially reduced if the methodis used in large scale production of the substrates. Moreover, thechemical linker between the substrate such as a solid support and thefunctional substance is non-hydrolyzable. More advantageously, theinertness of the linker also attributes stability to the immobilizedfunctional substance as the possibility of linker breakage due toundesirable chemical reactions is reduced.

The disclosed method also enables the user to vary the distance of theactive oxirane groups from the substrate. When the active oxirane groupsare at a suitable distance from the substrate, their reactivity towardsthe immobilization of functional substances may increase due to reducedsteric hindrance. In addition, the linker may be chosen to ensure a highloading of reactive epoxide groups, which in turn translates to a highloading of functional substances. More advantageously, the linker mayalso be chosen such that it inherently possesses certain desiredchemical properties. For example, when di-amine linkers are chosen, thefinal substrate obtained may have an inherent pH-buffering property.This is especially useful in applications like peritoneal dialysis wherethe lifespan and the efficacy of the sorbent may be adversely affectedby a high or low pH.

The method also allows the easy post-assembly modification ofoff-the-shelf dialysis membranes such as cellulose-acetate baseddialysers with urease. Urease can be immobilised after assembly, and canalso be immobilised on one face of the membrane only.

The substrate obtained from the disclosed method also allowsimmobilization of the biological substance thereon to be carried out ina simple, robust and user friendly way. For example, the immobilizationof a biological substance can be easily carried out at a laboratorylevel. This is so, because the immobilization of the biologicalsubstance can be carried out at ambient temperatures (e.g. roomtemperatures) in water/buffer solutions without requiring additionalchemicals or reagents. Advantageously, the absence of additionalchemicals or reagents significantly facilitates the purification of theimmobilized product. The immobilized functional material obtained fromthe disclosed method can also be non-toxic, biodegradable andbiocompatible. Advantageously, this allows the substrate to be used formedical applications, such as for example in dialysis applications as asorbent to remove unwanted waste products from the human body.Furthermore, these properties also allow the product to be used inenvironmental applications such as water treatment, soil treatment, orwaste treatment.

In addition, the disclosed method and substrate may also be useful inany one of the following applications: affinity chromatography, solidphase materials for chromatography (chiral), molecular imprinting,immobilizing dyes, sensors, biosensors, organic filters for selectivetoxin absorption, pharmaceutical applications (coating and binding),solid phase ion exchangers, solid phase metal scavengers andanti-oxidants.

While reasonable efforts have been employed to describe equivalentembodiments of the present invention, it will be apparent to the personskilled in the art after reading the foregoing disclosure, that variousother modifications and adaptations of the invention may be made thereinwithout departing from the spirit and scope of the invention and it isintended that all such modifications and adaptations come within thescope of the appended claims.

1-21. (canceled)
 22. A method of immobilizing a functional molecule on asubstrate, wherein said substrate comprises compounds disposed thereoncoupled to a functional molecule, each compound having a chaincomprising: a moiety R that is chemically coupled to the substrate, saidmoiety R being selected from the group consisting of an ether, ester,carbonyl, carbonate ester, thioether, disulfide, sulfonyl, sulfonyl,carbonothioyl, amine, amide, carbamide, ureas and guanidines; and anepoxide-containing moiety that is coupled to the moiety R by a linkercomprising at least one nucleophilic group selected from the groupconsisting of an amine, hydroxyl and thiol; wherein the functionalmolecule comprises an enzyme selected from the group consisting ofurease, uricase, creatininase, lipases, esterases, cellulases, amylases,pectinases, catalases, acylase, catalase, esterase, penicillin amidase,and proteinase-K.
 23. The method as claimed in claim 22, furthercomprising the step of applying a substantially homogenous mixture ofstabilizing additives to the surface of the substrate wherein saidadditives are selected to stabilize said functional molecule.
 24. Themethod as claimed in claim 23, wherein the step of applying thesubstantially homogenous mixture of additives comprises evaporating asolvent of a solution of said additives onto the substrate.
 25. Themethod as claimed in claim 23, wherein the stabilizing additives areselected from the group consisting of a sugar, an organic acid, an aminoacid, a sugar acid and a thiol.
 26. A method of preparing a substratecomprising immobilized functional molecules, the method comprising thesteps of: (i) providing electrophilic compounds coupled to the surfaceof the substrate; (ii) allowing the electrophilic compounds to undergo anucleophilic substitution reaction to provide a nucleophilic groupthereon and thereby increase the nucleophilicity of the substratesurface; (iii) allowing the nucleophilic group to undergo a nucleophilicsubstitution reaction with another electrophilic compound to provide anelectrophilic group on the substrate surface and thereby increase theelectrophilicity of the substrate; each electrophilic compound from step(iii) being coupled to said functional molecules, wherein the functionalmolecules comprise enzymes selected from the group consisting of urease,uricase, creatininase, lipases, esterases, cellulases, amylases,pectinases, catalases, acylase, catalase, esterase, penicillin amidase,and proteinase-K.
 27. The method as claimed in claim 26, wherein steps(ii) and (iii) are repeated n number of times to form n generations ofelectrophilic groups on said substrate
 28. The method as claimed inclaim 26, wherein said step of providing electrophilic compounds coupledto the surface of the substrate comprises chemically coupling a firstelectrophilic compound to the substrate.
 29. The method as claimed inclaim 28, wherein said step (ii) comprises the step of reacting anucleophile with the first electrophilic compound.
 30. The method asclaimed in claim 29, wherein step (iii) comprises chemically coupling asecond electrophilic compound to the nucleophile.
 31. The method asclaimed in claim 26, wherein the electrophilic compound is anepoxide-containing compound.
 32. The method as claimed in claim 31,wherein the epoxide-containing compound is epihalohydrin.
 33. The methodas claimed in claim 26, wherein the nucleophile is a di-nucleophile. 34.The method as claimed in claim 33, wherein the nucleophile comprises anamine.
 35. The method as claimed in 34, wherein the amine is selectedfrom the group consisting of saturated and unsaturated aliphatic oraromatic amines and polyamines.
 36. The method as claimed in claim 35,wherein the aliphatic group of said amines and polyamines is selectedfrom an alkyl group.
 37. The method as claimed in 36, wherein thepolyamine is selected from at least one of ethane-diamine,propane-diamine, butane-diamine, pentane-diamine, hexane-diamine. 38.The method as claimed in claim 26, wherein the substrate comprises apolymer.
 39. The method as claimed in claim 38, wherein the polymer is abiocompatible polymer.
 40. The method as claimed in claim 39, whereinthe biocompatible polymer is selected from the group consisting of apolyester substrate, a polyamide substrate, a polyacrylate substrate,and a polysaccharide-based substrate.
 41. The method as claimed in claim40, wherein the polysaccharide-based substrate is selected from thegroup consisting of cotton linters, cotton pulp, cotton fabrics,cellulose fibers, cellulose beads, cellulose powder, microcrystallinecellulose, cellulose membranes, rayon, cellophane, cellulose acetate,cellulose acetate membranes, chitosan, chitin, dextran derivatives andagarose derivatives.
 42. The method as claimed in claim 41, wherein thepolymer is a biopolymer.
 43. The method as claimed in claim 26, furthercomprising the step of applying a substantially homogenous mixture ofstabilizing additives to the surface of the substrate wherein saidstabilizing additives are selected to stabilize said functionalmolecule.
 44. The method as claimed in claim 43, wherein the step ofapplying the substantially homogenous mixture of additives comprisesevaporating a solvent of a solution of said additives onto thesubstrate.
 45. The method as claimed in claim 43, wherein thestabilizing additives are selected from the group consisting of a sugar,an organic acid, an amino acid, a sugar acid and a thiol. 46-49.(canceled)
 50. A method of modifying a dialysis membrane comprisingimmobilized functional molecules, the method comprising the steps of:(i) coupling electrophilic compounds to the membrane surface; (ii)allowing the electrophilic compounds to undergo a nucleophilicsubstitution reaction to provide a nucleophilic group thereon andthereby increase the nucleophilicity of the membrane surface; and (iii)allowing the nucleophilic group to undergo a nucleophilic substitutionreaction with another electrophilic compound to provide an electrophilicgroup on the membrane surface and thereby increase the electrophilicityof the membrane surface for immobilizing functional molecules thereon;each electrophilic compound from step (iii) being coupled to thefunctional molecules, wherein the functional molecules comprise enzymesselected from the group consisting of urease, uricase, creatininase,lipases, esterases, cellulases, amylases, pectinases, catalases,acylase, catalase, esterase, penicillin amidase, and proteinase-K.
 51. Amethod, as claimed in claim 50, wherein said membrane is a celluloseacetate membrane.